Abstract

   Rabbits are a suitable animal model for atherosclerosis due to their
   sensitivity to dietary cholesterol. Moreover, rabbits have lipoprotein
   profiles that are more similar to humans than those of other laboratory
   animals. However, little is known about the transcriptomic information
   related to atherosclerosis in rabbits. We aimed to determine the
   changes in the livers of rabbits fed a normal chow diet (control) or
   high cholesterol diet (HCD) by histological examinations and RNA
   sequencing analysis. Compared with the control group, the lipid levels
   and small LDL subfractions in plasma were increased, and aortic
   atherosclerotic plaques were formed in the HCD group. Most importantly,
   HCD resulted in lipid accumulation and inflammation in the livers.
   Transcriptomic analysis of the liver showed that HCD induces 1183
   differentially expressed genes (DEGs) that mainly participate in the
   regulation of inflammation and lipid metabolism. Furthermore, the
   signaling pathways involved in inflammation and lipid metabolism were
   enriched by KEGG pathway analysis. In addition, hepatic DEGs of the HCD
   group were further validated by real-time PCR. These results suggest
   that HCD causes liver lipid accumulation and inflammatory response.
   Although the relationships between these hepatic changes and
   atherogenesis need further investigation, these findings provide a
   fundamental framework for future research on human atherosclerosis
   using rabbit models.

Introduction

   Atherosclerosis is a complex multifactorial disease of the aorta and
   muscular arteries and the leading cause of morbidity and mortality
   throughout the world^[40]1. Many laboratory animal models have been
   used to study the process of atherogenesis, including mice, rabbits,
   pigs and nonhuman primates. In particular, mouse models are widely used
   due to the relatively short time frame for the progression of
   atherosclerosis and the relative ease of genetic manipulation^[41]2.
   While not all aspects of mouse atherosclerosis are identical to humans,
   several characteristics of lipid metabolism in rabbits make them
   particularly suitable for the study of human hypercholesterolemia and
   atherosclerosis. First, a large body of evidence suggests that high
   levels of plasma cholesterol, especially low-density lipoprotein
   (LDL)-cholesterol, result in atherosclerotic lesion formation in
   humans^[42]3. Unlike mice, in which high-density lipoproteins (HDLs)
   are the predominant plasma lipoproteins, rabbit lipoprotein profiles
   are LDL-rich, similar to those of humans^[43]4,[44]5. Second, it has
   been reported that cholesteryl ester transfer protein (CETP) plays a
   central role in lipid metabolism and atherosclerotic processes^[45]6.
   Rabbits have abundant CETP activity in the plasma as do humans, whereas
   mice do not have an endogenous CETP gene^[46]7,[47]8. Third, rabbits do
   not have hepatic ApoB mRNA editing activity as humans, so rabbit
   ApoB-48 is only present in chylomicrons as in humans. However, ApoB-48
   is present in all ApoB-containing particles such as very low-density
   lipoproteins (VLDLs), LDLs and chylomicrons in mice^[48]9. In addition,
   hepatic lipase (HL), which plays a major role in lipoprotein
   metabolism, is bound to the vascular endothelium in rabbits and humans,
   whereas the majority of HL is circulating in the plasma in
   mice^[49]10,[50]11. Therefore, rabbits provide a unique system to study
   the initiation and progression of atherosclerosis in humans. Despite
   the importance of rabbit models for the study of human
   hypercholesterolemia and atherosclerosis, little is known about the
   transcriptomic information related to atherosclerosis in rabbits.

   Next-generation sequencing platforms have made genomic and
   transcriptomic analyses affordable for researchers to elucidate the
   molecular mechanisms of human diseases. RNA sequencing (RNA-Seq) uses
   massively parallel sequencing to analyze the transcriptome at higher
   resolution than Sanger sequencing- and microarray-based
   methods^[51]12,[52]13. We envision that investigation of the
   transcriptomics in rabbits fed a high cholesterol diet can provide a
   better understanding of the biochemical and molecular processes
   involved in the initiation and progression of atherosclerosis in this
   model.

   Therefore, in the present study, we conducted RNA-Seq analysis of
   livers in rabbits fed a high cholesterol diet coupled with analyses of
   the plasma lipids and lipoprotein subfractions, aortic atherosclerotic
   lesions and liver pathology.

Results and Discussion

Plasma lipid levels and lipoprotein subfractions

   Compared with the normal chow diet (control) group, the plasma levels
   of total cholesterol (TC) and triglycerides (TG) were increased by
   19.4-fold and 1.7-fold in high cholesterol diet (HCD)-fed rabbits for
   16 weeks, respectively (Fig. [53]1, P < 0.01).

Figure 1.

   [54]Figure 1
   [55]Open in a new tab

   The plasma lipid levels in rabbits. The rabbits were fed a normal chow
   diet or 0.3% high cholesterol diet for 16 weeks. The plasma levels of
   TC and TG were measured. Data are expressed as the mean ± SEM. n = 10
   for each group. **P < 0.01 vs. the control group.

   As shown in Fig. [56]2, plasma lipoprotein subfraction analysis showed
   that the levels of VLDLs, intermediate-density lipoproteins (IDLs) and
   LDLs were markedly increased in the HCD group compared with the control
   group. It is well established that increased LDL levels are considered
   a major risk factor for atherosclerosis^[57]14,[58]15. However, an
   increasing number of studies have found that the disproportionate
   number of small and denser LDL particles is an independent risk factor
   for cardiovascular diseases because small LDL particles may reside
   longer in circulation and may be more prone to uptake by macrophages in
   atherosclerosis^[59]16,[60]17. Therefore, we further analyzed the size
   distribution of LDL particles using the Lipoprint LDL System. The
   system can resolve plasma lipoproteins to discrete bands consisting of
   VLDL; IDL bands A, B, and C; LDL subfractions 1 to 7. Subfraction 1
   represents large LDL particles, subfraction 2 indicates intermediate
   LDL particles, and subfractions 3–7 refer to small LDL
   particles^[61]18. We found that small LDL particles were basically not
   present in the control rabbits whereas they (fractions 3–4) were
   dramatically increased in the HCD-fed rabbits (Fig. [62]2C). It has
   been reported that small LDL particles are more atherogenic than large
   buoyant LDL particles^[63]19,[64]20. Recent clinical trials also showed
   that small LDL particles were markers of early atherosclerosis, and the
   measurement of small LDL particles provided important information in
   the risk assessment for atherosclerotic disease^[65]21,[66]22. In
   addition, there were obvious differences in the plasma levels of VLDL,
   IDL-C, IDL-B and IDL-A between the HCD group and the control group in
   our study (P < 0.01). Data from the present study showed that high
   dietary cholesterol increased the plasma levels of TC and TG, and
   increased plasma TC levels were mainly caused by elevated small LDL
   particles in rabbits.

Figure 2.

   Figure 2
   [67]Open in a new tab

   The plasma lipoprotein subfraction analysis in rabbits. Blood samples
   in the control group and HCD group were used for lipoprotein
   subfraction analysis. Data are expressed as the mean ± SEM. n = 3 for
   each group. *P < 0.05 and **P < 0.01 vs. the control group.

Gross and histological changes in atherosclerotic lesions

   Gross atherosclerotic lesions and histological features in the aorta
   were examined. Aortas of rabbits were collected and stained with Sudan
   IV. Compared with the control group, which contained no visible
   lesions, en face lesions in the HCD group were remarkable and
   distributed from the aortic arch, thoracic aorta to abdominal aorta
   with predominance in the aortic arch (Fig. [68]3A).

Figure 3.

   [69]Figure 3
   [70]Open in a new tab

   Gross and histological changes in atherosclerotic lesions in rabbits.
   (A) Representative pictures of Sudan IV staining in aortas. (B) The
   aortic atherosclerotic lesions were stained with H&E or EVG. (C) The
   aortic atherosclerotic lesions were stained with antibodies against MФ
   or SMC by immunohistochemistry. Bar = 200 μm.

   To further analyze the histological changes of atherosclerotic lesions,
   we examined the intimal lesions in the aortic arch using hematoxylin
   and eosin (H&E) and Elastica van Gieson (EVG) staining. We found that
   the intimal lesions were mainly composed of macrophage-derived foam
   cells, whereas no lesions were found in the control group
   (Fig. [71]3B). It is generally accepted that the enhancement of intimal
   macrophage (MФ) accumulation may increase the vulnerability of the
   plaques or make the plaques prone to rupture, leading to thrombosis in
   atherosclerosis^[72]23. In addition, vascular smooth muscle cell (SMC)
   proliferation also plays a crucial role in the progression of
   atherosclerosis^[73]24. Immunohistochemical staining showed that in
   addition to macrophages, the lesions also contained many SMCs
   (Fig. [74]3C). Thus, aortic atherosclerotic lesions were well formed in
   rabbits fed a 0.3% cholesterol diet for 16 weeks.

Histological changes in liver

   Accumulating evidence suggests that the liver plays a key role in the
   inflammatory response evoked by dietary cholesterol. Clinical studies
   found that liver-derived inflammation markers such as C-reactive
   protein and serum amyloid A (SAA) were rapidly increased after
   consumption of an excess amount of dietary cholesterol, thereby
   promoting the onset of early aortic lesion formation^[75]25,[76]26.
   These results indicated that liver plays a key role in the inflammatory
   response evoked by dietary constituents. We found that there were
   significant steatosis, ballooning and inflammation in the HCD group
   compared with the control group (Fig. [77]4A). In addition,
   histological scoring for inflammation was based on the Brunt
   classification^[78]27,[79]28. There was a trend toward higher
   inflammation in the HCD group compared with the control group
   (P < 0.01) (Fig. [80]4B). The results of Oil Red O revealed clear lipid
   accumulation and severe macrovesicular hepatic steatosis in the HCD
   group compared with the control group (Fig. [81]4C and D). These
   results suggest that HCD could cause fatty degeneration and
   inflammatory response in the liver. In support of our observations, it
   has been shown that dietary cholesterol induced pro-inflammatory gene
   expression in the livers of ApoE*3Leiden transgenic mice^[82]29.

Figure 4.

   [83]Figure 4
   [84]Open in a new tab

   Histological changes in the livers of rabbits by H&E and Oil Red O
   staining. (A) Compared to the control liver, the hepatocytes of HCD-fed
   rabbits show fatty degeneration and accumulation of mononuclear
   infiltration in the center (indicated by an arrow). (B) Hepatic
   inflammation was quantified using the Brunt classification. (C)
   Representative Oil Red O staining of livers in the control group and
   HCD group. (D) Oil Red O staining area was measured using the WinROOF
   image analysis software. Data are expressed as the mean ± SEM. n = 4
   for each group. **P < 0.01 vs. the control group. Bar = 100 μm.

RNA-Seq analysis of hepatic gene expression

   To gain insight into the complex traits underlying the
   pathophysiological response of the liver to dietary cholesterol,
   transcriptomic analysis was performed by RNA-Seq. In the previous
   study, we identified the gene mutations or modifiers through
   whole-genomic sequencing and deep transcriptome sequencing of LDL
   receptor deficient (WHHL) rabbits and cholesterol-fed New Zealand White
   rabbits. The previous study mainly addressed transcriptional changes in
   the WHHL rabbits, while cholesterol-fed New Zealand White rabbits were
   used as a reference^[85]30. To further emphasize the relationship with
   lipoprotein profiles and aortic atherosclerosis, in the current study,
   we focused on the hepatic gene expression changes and liver pathology
   in the setting of hepatic steatosis induced by cholesterol diet in the
   Japanese White rabbits. The transcriptomic analysis revealed that there
   were 1,183 differentially expressed genes (DEGs) compared with the
   control rabbits. Of them, 984 genes were up-regulated and 199 genes
   were down-regulated compared with the control group (Supplementary
   Table [86]S1). Kleemann et al. found that 1,896 genes were
   significantly changed in the livers of ApoE*3Leiden mice fed a
   cholesterol diet during atherogenesis by GeneChip microarrays^[87]29.
   To investigate the functions of these 1,183 DEGs, we grouped them into
   five categories according to their functions, including glucose
   metabolism, lipid metabolism, protein metabolism, cell
   proliferation/apoptosis and inflammation. Our results revealed that HCD
   predominantly affected genes involved in inflammation and lipid
   metabolism in the liver (Fig. [88]5 and Supplementary Table [89]S2). As
   shown in Fig. [90]5 and Table [91]S1, we found that the adaptation of
   hepatic lipid metabolism to dietary cholesterol was mainly controlled
   by the genes of cholesterol biosynthesis and lipid metabolism. Sterol
   regulatory element binding protein 1 (SREBF1), a key transcriptional
   factor in modulating cholesterol biosynthesis, was up-regulated
   40.7-fold compared to the control rabbits. Karasawa et al. revealed
   that hepatic SREBP1 determined plasma remnant lipoproteins and
   contributed to atherosclerosis in LDLR−/− mice^[92]31. It has been
   reported that 24-dehydrocholesterol reductase (DHCR24) is likely a
   target for SREBPs, which has been suggested to regulate the genes
   involved in cholesterol biosynthesis^[93]32. Our results showed that
   the DHCR24 gene was down-regulated 2.1-fold in HCD-fed rabbits.
   Furthermore, HCD induced many genes that mediate the inflammatory
   response in the liver. These genes include proteases (MMP2, MMP14,
   MMP15, TIMP1, TIMP2, TIMP3), chemokines (CCL25, CCL21, CXCL16, MIF),
   adhesion molecules (CD74, CD14, CD97, ICAM1) and cardiovascular risk
   factors (SAA3, CEBPB). It is generally accepted that atherosclerosis is
   a multifactorial and persistent condition and is regarded as a form of
   chronic inflammation induced by lipid accumulation^[94]3. Other studies
   showed that there is a link between cholesterol and inflammation in
   ApoE−/− mice and ApoE*3Leiden mice fed with high
   cholesterol^[95]29,[96]33. Taken together, these findings suggest that
   hepatic lipid metabolism and inflammation may be closely associated
   with the initiation and progression of atherosclerosis in rabbits.

Figure 5.

   Figure 5
   [97]Open in a new tab

   General features of differentially expressed genes in the livers of
   rabbits. The DEGs were analyzed using DESeq software by comparing the
   control and HCD groups. Expression intensities are displayed from blue
   (low expression) to red (high expression), and the expression level is
   proportional to the brightness of the color (see color bar). n = 3 for
   each group.

   The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis
   provided additional possible functional information showing the
   pathways relevant to DEGs during atherogenesis. In concordance with our
   DEG results, analysis of the KEGG pathway showed that these DEGs were
   mainly contained in lipid metabolism- and inflammation-related
   signaling pathways including PI3K-Akt, MAPK, ABC transporters, FoxO,
   PPAR, AMPK, NF-κB, mTOR, tumor necrosis factor (TNF), Toll-like
   receptor and cGMP-PKG signaling pathways. Significantly, HCD activated
   specific inflammatory pathways, such as NF-κB, Toll-like receptor and
   TNF signaling pathways in rabbits (Fig. [98]6 and Supplementary
   Table [99]S3). As shown in Fig. [100]6, the number and percentage of
   DEGs in these signaling pathways were analyzed. We found that the
   largest functional pathway was the MAPK signaling pathway, representing
   a total of 21 DEGs (~12% of the total). It has been reported that these
   enriched signaling pathways participate in the inflammatory response
   and lipid metabolism in atherosclerosis^[101]34,[102]35. Vergnes et al.
   also discovered that the hepatic inflammatory response was evoked by
   high cholesterol feeding in LDLR−/− mice^[103]36. These above studies
   suggest that the uptake of dietary cholesterol leads to the
   inflammatory response of the liver in rabbits.

Figure 6.

   [104]Figure 6
   [105]Open in a new tab

   Enriched KEGG pathways of differentially expressed genes in the livers
   of rabbits. The DEGs were mapped into the KEGG databases, significantly
   enriched KEGG terms were determined by P ≤ 0.05. n = 3 for each group.

Quantitative real-time PCR analysis

   Both hypercholesterolemia and inflammatory response are well-known risk
   factors for the development of atherosclerosis and have been shown to
   play a causal role in the progression of atherosclerotic
   plaques^[106]3. To validate the DEGs in the liver, real-time PCR was
   carried out. As shown in Fig. [107]7, the genes SREBF1, LDLR and CYP7A1
   involved in lipid metabolism were significantly changed in the livers
   of the HCD group compared with the control group. Furthermore, we found
   that the expression levels of inflammatory genes MMP2, TIMP1, MIF,
   ICAM1 and SAA3 were increased, whereas the expression level of
   interleukin-1R2 (IL-1R2) was decreased. Previous studies showed that
   the proinflammatory cytokine IL-1 interacts with cells through two
   types of receptors, type I receptor (IL-1R1) and IL-1R2. IL-1R2
   inhibits IL-1 activity by acting as a decoy target for
   IL-1^[108]37,[109]38. These results indicate that dietary cholesterol
   can be an important trigger and a possible source of the inflammatory
   component.

Figure 7.

   Figure 7
   [110]Open in a new tab

   Quantitative real-time PCR verification. Real-time PCR was performed
   essentially to validate the DEGs in the livers of the control group and
   HCD group. Data are expressed as the mean ± SEM. n = 3 for each group.

   The correlation was calculated to compare the expression levels between
   RNA-Seq and real-time PCR. Fold changes in DEGs between the two
   techniques were significantly correlated in the depot (Pearson’s
   R = 0.88) (Fig. [111]8). The results confirmed that these DEGs
   identified in our study were very reliable.

Figure 8.

   Figure 8
   [112]Open in a new tab

   The correlation of log2-fold change in gene expression in the liver of
   rabbits between the RNA-Seq and real time-PCR.

Conclusions

   The results from the current study demonstrate that dietary cholesterol
   is not only a risk factor for hypercholesterolemia but also a trigger
   of hepatic inflammation. Although the significance of hepatic
   inflammation in the development of atherosclerosis deserves further
   investigation, these results provide valuable information for the study
   of both hypercholesterolemia and hepatic inflammation using rabbit
   models. Further studies are ongoing to validate these findings.

Materials and Methods

Animals and diets

   Male Japanese White rabbits (2.5–3.0 kg) were provided by the
   Laboratory Animal Center of Xi’an Jiaotong University. In the current
   study, we aimed to determine the influence of hypercholesterolemia on
   transcriptomic change and liver pathology that may be related to
   atherosclerosis in rabbits. It is well known that rabbits are sensitive
   to dietary cholesterol and rapidly develop severe hypercholesterolemia
   leading to atherosclerosis^[113]5. However, rabbits are out-bred and
   show a wide biological variability in terms of individual
   responsiveness to dietary cholesterol^[114]39. To minimize the
   intra-group variations of transcriptomic analysis caused by different
   response to a cholesterol diet, thirty rabbits were fed a chow diet
   containing 0.5% cholesterol for one week. To ensure that all rabbits
   had a similar responsiveness to cholesterol feeding, we selected
   rabbits based on the plasma levels of TC (ranging from 300 to
   500 mg/dl). Eventually, twenty rabbits were selected and subsequently
   divided into two groups: normal chow diet group and cholesterol diet
   group fed a 0.3% cholesterol diet. All rabbits were given a restricted
   diet of 100 g/day for 16 weeks. Rabbits were individually housed in
   metal cages in air-conditioned rooms under a 12 h light/12 h dark cycle
   and were given free access to water throughout the experiments. The
   experimental protocol was in accordance with the National Institutes of
   Health Guide for Care and Use of Laboratory Animals and was approved by
   the Laboratory Animal Care Committee of Xi’an Jiaotong University.

Biochemical analysis

   Blood samples were collected from the ear artery of rabbits after
   fasting for 16 h. The plasma levels of TC and TG were measured using
   commercial kits (Biosino Bio-technology and Science, Beijing, China).
   Plasma samples were also used for lipoprotein subfraction analysis. The
   analysis was performed electrophoretically using high-resolution 3%
   polyacrylamide gel tubes and the Lipoprint LDL System (Quantimetrix
   Corporation, Redondo Beach, CA, USA) according to the manufacturer’s
   instructions as previously described^[115]40,[116]41.

Determination of atherosclerotic lesions

   Rabbits were sacrificed with an injection of phenobarbital sodium and
   xylazine hydrochloride. The aortic tree was isolated, opened
   longitudinally, fixed in 10% neutral buffered formalin, and stained
   with Sudan IV^[117]42. Then, the aortic arch was cut into 10 sections.
   The sections were embedded in paraffin and cut into 5-μm thick serial
   sections, stained with H&E and EVG. For the evaluation of cellular
   components in atherosclerotic lesions, the sections were
   immunohistochemically stained with antibodies against RAM11 (macrophage
   marker) (Dako, Carpinteria, CA, USA) and HHF35 (SMC marker) (Thermo,
   Rockford, IL, USA).

Histology and Immunohistochemistry

   The livers were fixed in 10% neutral buffered formalin and processed
   for embedding in paraffin. Then, specimens were cut into 5-μm thick
   serial sections and stained with H&E. Frozen sections of liver (5-μm
   thick) were stained with Oil Red O for evaluation of hepatic fatty
   change. The Oil Red O staining area was measured using the WinROOF
   image analysis software (Mitani, Tokyo, Japan).

Total RNA extraction and cDNA library construction

   Total RNA was extracted from the liver using RNAzol (Takara, Tokyo,
   Japan). RNA concentrations and purity were determined using Nanodrop
   (Thermo, Rockford, IL, USA), and integrity was verified using an
   Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
   mRNA was purified from total RNA using the NEBNext® Poly(A) mRNA
   magnetic isolation module. A total of 6 paired-end libraries were
   constructed using a NEBNext UltraTM RNA Library Prep Kit following
   Illumina manufacturer’s recommendation. Library quality was assessed on
   the Agilent Bioanalyzer 2100 system. The clustering of the index-coded
   samples was performed on a cBot Cluster Generation System using TruSeq
   PE Cluster Kit v4-cBot-HS (Illumina). The libraries were sequenced on
   an Illumina HiSeq. 2500 platform with 100 bp paired-end reads
   (Illumina, San Diego, CA, USA).

Differentially expressed gene analysis

   The Perl script was used to trim the reads with contaminated adapters,
   more than 20% low-quality bases (Phred quality score < 20), and more
   than 10% Ns. First, we obtained clean reads (Supplementary
   Table [118]S4). Then, clean reads were aligned to the rabbit reference
   genome (GCA_000003625) using TopHat. The gene expression was quantified
   and normalized by Cufflinks in RPKM (reads per million per kilo
   bases)^[119]43. The DEGs were analyzed using DESeq software by
   comparing the control and HCD groups^[120]44. The significance
   threshold of the p-value in multiple tests was set by the false
   discovery rate (FDR). The threshold, the absolute values of fold
   change ≥ 2 and FDR < 0.05, was applied to judge the significance of
   gene expression. For the functional and pathway enrichment analysis,
   the DEGs were then mapped into the KEGG databases, and significantly
   enriched KEGG terms were determined by P ≤ 0.05.

Real-time PCR

   Real-time PCR was performed essentially to validate and confirm the
   differences in gene expression between the HCD group and the control
   group. Total RNA was extracted from the liver using RNAzol. cDNAs were
   synthesized using a RevertAid First Strand cDNA Synthesis Kit (Takara,
   Tokyo, Japan) following the manufacturer’s instructions. Real-time PCR
   was performed with the SYBR Green PCR master mix (Takara, Tokyo, Japan)
   using a thermal cycler dice real time system. Primers were designed
   with primer premier 5.0 (Premier Biosoft, Palo Alto, CA, USA)
   (Supplementary Table [121]S5). GAPDH was used as an endogenous control.
   The relative gene expression was quantitatively analyzed by the
   comparative Ct method (2^−ΔΔCT). Data were normalized to rabbit GAPDH
   mRNA levels.

Statistical analysis

   Data are presented as the mean ± SEM. Statistical analysis was
   performed by the two-tailed Student’s t-test using GraphPad Prism
   software. Differences between groups were considered statistically
   significant if P < 0.05.

Electronic supplementary material

   [122]Table S1^ (128.5KB, xlsx)
   [123]Table S2^ (33KB, doc)
   [124]Table S3^ (23.5KB, xlsx)
   [125]Table S4^ (32KB, doc)
   [126]Table S5^ (36KB, doc)

Acknowledgements