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Acute myocardial infarction and stroke occur more frequently in the morning, suggesting a role of the circadian clock in these main causes of death, secondary to atherosclerosis.
To investigate the expression of clock genes, apoptosis-related genes and atherosclerosis-related genes in the process of atherosclerosis.
Apolipoprotein E knockout (ApoE−/−) mice were used to establish animal models of early and advanced atherosclerosis. Real-time polymerase chain reaction, Western blotting and microarray assays were used to detect the expression of clock genes, apoptosis-related genes and atherosclerosis-related genes.
Clock genes in ApoE−/− and C57BL/6J mouse hearts exhibited daily oscillations at the messenger RNA level. However, the expression level and rhythm between ApoE−/− and C57BL/6J mice were significantly different. Moreover, the changes became more significant as atherosclerosis developed. c-Myc and p53 genes exhibited circadian expression in C57BL/6J mice at messenger RNA and protein levels. However, the rhythm in ApoE−/− mice disappeared completely. Bcl-2 and Bax did not show daily rhythm in either strain of mouse. Aside from apoptosis-related genes, several atherosclerosis-related genes expressed time-dependent behaviour in C57BL/6J mice but not in ApoE−/− mice.
Rhythm changes of clock genes, apoptosis-related genes and atherosclerosis-related genes may play important roles in atherosclerosis and its complications.
Les infarctus aigus du myocarde et les accidents vasculaires cérébraux se produisent surtout le matin, ce qui laisse supposer que l’horloge circadienne joue un rôle dans ces importantes causes de décès imputables à l’athérosclérose.
Explorer l’expression des gènes de l’horloge interne, des gènes liés à l’apoptose et des gènes liés à l’athérosclérose dans le processus d’athérosclérose.
Les auteurs ont utilisé des souris dont l’alipoprotéine E est inactivée (ApoE/−/−) pour établir des modèles animaux d’athérosclérose précoce et avancée. Ils ont recouru à la réaction en chaîne de la polymérase en temps réel, au transfert Western et aux titrages de microréseau pour déceler l’expression des gènes de l’horloge interne, des gènes liés à l’apoptose et des gènes liés à l’athérosclérose.
Les gènes de l’horloge interne du cœur de souris ApoE/−/− et C57BL/6J affichaient des oscillations quotidiennes dans l’ARN messager. Cependant, le taux d’expression et le rythme entre les souris ApoE/−/− et C57BL/6J différaient considérablement. De plus, ces changements sont devenus encore plus importants avec l’évolution de l’athérosclérose. Les gènes c-Myc et p53 présentaient une expression circadienne de l’ARN messager et des protéines chez les souris C57BL/6J. Cependant, le rythme des souris ApoE/−/− a complètement disparu. Les gènes Bcl-2 et Bax n’ont pas démontré de rythme quotidien dans l’une ou l’autre souche de souris. En plus des gènes liés à l’apoptose, plusieurs gènes liés à l’athérosclérose ont exprimé un comportement en fonction du temps chez les souris C57BL/6J, mais pas chez les souris ApoE/−/−.
Les changements de rythme des gènes de l’horloge interne, des gènes liés à l’apoptose et des gènes liés à l’athérosclérose jouent peut-être un rôle important dans l’athérosclérose et ses complications.
Many aspects of cardiovascular function are subject to diurnal variation, although the precise role of the circadian clock in these phenomena remains largely undetermined (1). Acute cardiovascular events exhibit an obvious circadian rhythm in the frequency of occurrence (2). One large-scale multicentre study demonstrated that acute myocardial infarction is 1.28 times more likely to occur between 06:00 and 12:00 than during the other three 6 h intervals of the day (3). Pathological studies have revealed that acute myocardial infarction is the main cause of death in patients with atherosclerotic disease (4,5). The mechanisms of the phenomena are not yet fully understood, but the circadian clock and clock genes may be involved in the process.
It has been shown that the circadian clock has important roles in orchestrating temporal integration of physiology and behaviour with the environment. This master clock, which in mammals, resides in the hypothalamic suprachiasmatic nucleus, synchronizes multiple peripheral oscillators to ensure temporally coordinated physiology (6–8). Clocks outside the brain have been demonstrated by the measurement of free-running rhythms of gene expression in cultured peripheral tissues (9,10). Peripheral clocks also play important roles in physiological function regulation and in pathological situations including cardiovascular diseases. Studies have shown that clock genes function as tumour suppressors by regulating the expression of apoptosis-related genes. Atherosclerosis shares common pathogenetic mechanisms with cancer, and abnormal cell proliferation and apoptosis are also observed in atherosclerosis (11,12). Besides apoptosis-related genes, many other atherosclerosis-related genes are involved in the disease process. Thus, abnormal expression of clock genes and the corresponding disturbances of apoptosis-related and atherosclerosis-related genes may exist in the process of atherosclerosis. These abnormalities may, in turn, affect the development of the disease.
In the present study, an atherosclerotic model was established using apolipoprotein E knockout (ApoE−/−) mice. The expression profiles of clock genes, apoptosis-related genes and atherosclerosis-related genes in ApoE−/− mice and C57BL/6J control mice were examined. Our data may provide new insights to elucidate the role of the circadian clock in atherosclerosis.
Forty-eight male ApoE−/− mice and 24 male C57BL/6J control mice (12 weeks of age) were used in the study. ApoE−/− and C57BL/6 mice were purchased from the Peking University Health Science Center (Beijing, China) and the Shanghai Laboratory Animal Center, Chinese Academy of Science (Shanghai, China), respectively. All procedures performed were in accordance with institutional policies. Animals were maintained on a light-dark cycle (12 h of light and 12 h of dark) at a temperature of 23°C for four weeks. ApoE−/− mice were divided into two groups (24 mice each). ApoE−/− mice in the first group were fed a Western-type diet containing 0.15% cholesterol and 21% fat. ApoE−/− mice in the second group were fed normal standard rodent chow, as were C57BL/6J control mice.
According to Zeitgeber time (ZT) (ZT0 is defined as lights on) (13), mice were anesthetized at different time points including ZT0, ZT4, ZT8, ZT10, ZT12, ZT14, ZT16 and ZT20. After anesthetization, blood was obtained for serum preparation by removing the eyeballs of the mice. The levels of serum total cholesterol, low-density lipoprotein- bound cholesterol (LDL-C) and high-density lipoprotein-bound cholesterol (HDL-C) were measured using kits obtained from Shanghai Rongsheng Biotech Co, Ltd (China). The hearts and aortic intimae were rapidly isolated, frozen in liquid nitrogen and stored at −80°C until RNA and protein isolation. The arch of each aorta was also removed and stored in buffered formalin (10%). After dehydration in a 20% sucrose solution, the aortic segments were embedded in optimal cutting temperature compound. Frozen serial cross-sections (10 μm in thickness) were prepared for staining. Lillie-Ashburn’s oil red O staining method was used to detect atherosclerotic plaques.
Total cardiac RNA was prepared using TRIzol reagent (Invitrogen Corporation, USA), according to manufacturer instructions. The purity and integrity of RNA were assessed spectroscopically and by gel electrophoresis before use. Single-strand complementary DNA was prepared from 2 μg of total RNA following the protocol of the RevertAid First Strand Synthesis Kit (Fermentas MBI, USA) for real-time polymerase chain reaction (PCR).
The messenger RNA (mRNA) levels of five clock genes – mPer1 (GenBank accession #NM_011065), mPer2 (GenBank accession #NM_011066), mClock (GenBank accession #NM_007715), mCry1 (GenBank accession #NM_007771) and mBmal1 (GenBank accession #NM_007489) – and four apoptosis-related genes – c-Myc (GenBank accession #NM_010849), p53 (GenBank accession #NM_011640), Bcl-2 (GenBank accession #NM_009741) and Bax (GenBank accession #NM_007527) – were detected by real-time PCR (iCyler iQ Real-time PCR Detection System, Bio-Rad Laboratories Inc, USA). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (GenBank accession #NM_001001303) was used as an internal standard. Primer pairs were designed according to the published data of these genes in GenBank. The sequences of the primers and their annealing temperatures are shown in Table 1. PCR reactions were performed in a volume of 25 μL containing oligonucleotide primers (200 nM of each) and SYBR Green Realtime PCR Master Mix (Toyobo Co Ltd, Japan) (14). Melt analysis was performed to determine the specificity of the PCR amplification. PCR efficiency was identified by the slope of a standard curve.
Protein levels of the apoptosis-related genes c-Myc and p53 were determined by Western blot. Briefly, 30 μg of total protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked in phosphate-buffered saline containing 5% nonfat milk and 0.05% Tween-20 (Sigma-Aldrich Inc, USA) for 2 h and then incubated with primary antibody at 4°C overnight. The mouse monoclonal anti-c-Myc antibody and rabbit polyclonal anti-p53 antibody (Santa Cruz, USA) were diluted to 1:400 and 1:200, respectively. After washing in phosphate-buffered saline, the membranes were incubated with peroxidase-conjugated secondary antibodies at 37°C for 30 min. The protein bands of interest were detected by enhanced chemiluminescence (Pierce, USA). Protein bands of genes that varied by more than twofold in intensity were considered to be significantly different.
The hearts and aortic intimae of C57BL/6J mice and ApoE−/− mice fed the Western-type diet were collected at ZT0 and ZT12, and were used to determine the expression profile of atherosclerosis-related genes by microarray. The Oligo GEArray Atherosclerosis Microarray (OMM-038; SuperArray Bioscience Corporation, USA) profiles the expression of 113 key genes involved in atherosclerosis. All procedures were performed according to the manufacturer’s instructions. The expression levels were determined by a chemiluminescent detection kit (SuperArray Bioscience Corporation). The Internet-based, completely integrated GEArray Expression Analysis Suite (SuperArray Bioscience Corporation) was used to complete data analysis. Expression levels that varied by twofold or greater were considered to be significantly different.
Data are reported as mean ± SD. Data were subjected to the Student’s t test or ANOVA, and were considered to be significantly different if P<0.05.
No obvious atherosclerotic lesions formed in ApoE−/− mice fed with normal standard chow, but foam cells were observed under the endothelium (Figures 1A and 1B). After feeding on a Western-type diet for four weeks, atherosclerotic lesions formed and frozen-section analysis showed obvious lipid staining at the base of the aorta in ApoE−/− mice (Figure 1C). No lesions formed in C57BL/6J control mice (Figure 1D).
The total and LDL-C levels of the two groups of ApoE−/− mice were significantly higher than those of the C57BL/6J mice. No significant differences were found between the two groups of ApoE−/− mice. In addition, both groups of ApoE−/− mice showed lower HDL-C levels than control mice, and ApoE−/− mice fed with the Western-type diet had much lower HDL-C levels than those fed with normal chow (Table 2).
The mRNA levels of the clock genes mPer1, mPer2, mClock, mCry1 and mBmal1 in C57BL/6J and ApoE−/− mouse hearts were measured during a circadian cycle. All of the clock genes showed a clear daily rhythm at the mRNA level, except mClock (assessed by one-way ANOVA). However, it was found that the expression levels of clock genes in ApoE−/− mice were higher at most time points, with enhanced amplitudes and altered rhythms, than those in C57BL/6J control mice. These types of clock gene disturbances were more severe in ApoE−/− mice fed the Western-type diet than those fed with normal chow (Figure 2).
In C57BL/6J mice, mPer1 exhibited a daily rhythm with a peak at ZT10 and a trough at ZT20. ApoE−/− mice fed normal chow had a similar mPer1 rhythm but with a higher peak necessary. In ApoE−/− mice fed the Western-type diet, mPer1 showed a delayed peak and trough at ZT12 and ZT0, respectively (Figure 2A). The circadian rhythm of mPer2 in ApoE−/− mice fed the Western-type diet was similar to the C57BL/6J mice with the peak at ZT12, but its expression level was significantly elevated with enhanced amplitude. In ApoE−/− mice fed normal chow, the peak and the trough of mPer2 were at ZT10 and ZT0, respectively. In addition, the expression level was elevated but not as high as in ApoE−/− mice fed the Western-type diet (Figure 2B). In ApoE−/− mice fed the Western-type diet, mBmal1 mRNA expression showed a delayed peak at ZT4 with enhanced amplitude and higher expression level, whereas in C57BL/6J mice, the peak was at ZT0. In ApoE−/− mice fed normal chow, the circadian rhythm was similar to C57Bl/6J mice but with a higher expression level and similar amplitude (Figure 2C). mClock expression did not show any significant daily changes between the two strains of mice. The expression levels were higher in ApoE−/− mice, and ApoE−/− mice fed the Western-type diet had the highest mClock level (Figure 2D). The rhythm of mCry1 also changed in ApoE−/− mice: the peak was at ZT16 (16 h delayed compared with C57BL/6J mice) and the trough was at ZT4 (6 h advanced rather than delayed). The expression levels were elevated in the two groups of ApoE−/− mice, and ApoE−/− mice fed normal chow showed a lower expression level (Figure 2E). Thus, the changes of clock gene expression in ApoE−/− mice tended to become more severe with the development of atherosclerosis.
The daily expression of apoptosis-related genes in C57BL/6J and ApoE−/− mice was examined to detect the possible relationships of clock genes, apoptosis-related genes and atherosclerosis. The genes examined by real-time PCR included c-Myc, p53, Bcl-2 and Bax. It was found that c-Myc and p53 expression showed significant circadian changes at the mRNA level in C57BL/6 mice (assessed by one-way ANOVA). The c-Myc mRNA expression reached the peak at ZT20 and the trough at ZT14. For p53, the expression peak and trough were found at ZT0 and ZT12, respectively. However, the c-Myc and p53 mRNA expression did not show any daily oscillations in the two groups of ApoE−/− mice, and the expression levels in the two groups of ApoE−/− mice did not show significant differences (Figures 3A and 3B). Neither Bcl-2 nor Bax exhibited circadian variation in C57BL/6J and ApoE−/− mice (Figures 3C and 3D).
Similarly, protein expression of both c-Myc and p53 genes showed circadian rhythms in C57BL/6J mouse hearts. Figure 4 shows that the c-Myc protein expression increased gradually during the day and peaked at ZT12, then decreased until reaching the trough at ZT20. The time of the lowest level of p53 protein was ZT0 and then the level kept increasing until it reached the peak at ZT14. In the two groups of ApoE−/− mice, the expression levels of p53 and c-Myc proteins showed no significant rhythms during a circadian cycle.
Microarray was used to detect the expression profiles of 113 atherosclerosis-related genes at ZT0 and ZT12 in mouse hearts and aortic intimae. The expression levels of these genes were compared between the two time points. The results showed that some genes (20 in the heart and 15 in the aortic intimae) exhibited significant expression differences between ZT0 and ZT12 in C57BL/6J mice (Tables 3 and and4).4). Among them, only three genes were shared by the two tissue types: ACE, ApoA1 and MMP3. The expression levels of these genes changed with time, suggesting that they may be regulated by the circadian clock and are classified as clock-controlled genes (CCGs). Moreover, C57BL/6J mice had many more time-dependent genes than ApoE−/− mice. In ApoE−/− mice, there were only six and three time-dependent genes, respectively. Most time-dependent genes lost these trends in ApoE−/− atherosclerotic mice (15 genes in the heart and 12 in the aortic intimae) (Table 3). Only a few genes kept the time-dependent manner in both C57BL/6J and ApoE−/− mice: Bid, Ccr2, Fga, TGFβ3 and VEGFα in the heart, and CD36, ICAM2 and NFκB1 in the aortic intimae.
As a commonly used atherosclerotic mouse model, atherosclerotic lesions develop in ApoE−/− mice fed either normal chow or a Western-type diet. For ApoE−/− mice fed with a normal diet, lesions begin at five to six weeks of age, with monocyte attachment to the endothelium in lesion-prone areas and transendothelial migration, whereas obvious fibrous plaques appear after 20 weeks (15). In our study, after being fed the Western-type diet for four weeks, obvious plaques formed in the aortae of 16-week-old ApoE−/− mice. Meanwhile, after four weeks, obvious plaques did not form in ApoE−/− mice fed with normal chow, but foam cells were observed under the endothelium. The cholesterol and LDL-C levels did not show significant differences between the two groups, but ApoE−/− mice fed the Western-type diet had much lower HDL-C levels. Thus, the ApoE−/− mice fed with normal chow were at the early stage of atherosclerosis, while mice fed with the Western-type diet were already at the advanced stage.
Circadian rhythms are generated by the feedback loops of the core circadian genes (16). The circadian rhythms serve to organize many physiological functions (17). Recent studies showed that circadian variation is also related to diseases (18). In some cardiovascular diseases, circadian expression of clock genes may change (19,20). Our results showed that the clock genes exhibited daily oscillations at the mRNA level (except mClock) in C57BL/6J mice, as previously reported (21). However, clock genes in ApoE−/− mice showed increased expression levels, enhanced amplitudes and different circadian rhythms. Interestingly, abnormities of circadian genes were more severe in ApoE−/− mice at the advanced stage of atherosclerosis than in mice at the early stage. These results suggest that atherosclerosis may affect the expression of circadian genes, as with the other cardiovascular diseases mentioned above (19,20). Aside from clock genes, expression of many other genes show circadian rhythms (22). These CCGs are regulated by clock genes and the circadian clock controls physical functions through them. Thus, the expression changes of clock genes in ApoE−/− atherosclerotic mice will influence the expression of CCGs, which may, in turn, affect the process of atherosclerosis.
The circadian clock has been shown to affect cell growth and proliferation in several studies. The core circadian genes are involved in controlling the expression of cell cycle genes and tumour suppressor genes at the transcriptional and post-transcriptional levels (16). Fu et al (23) found that Per2 could function as a tumour suppressor gene by regulating p53 and c-Myc protein levels. Among the four apoptosis-related genes in our study (p53, Bcl-2, Bax and c-Myc), Bcl-2 and Bax did not show circadian rhythms in both ApoE−/− and C57BL/6J mice, suggesting that these genes may not be regulated directly by the circadian clock. In contrast, a daily rhythm of c-Myc expression was observed in the hearts of C57BL/6J mice. c-Myc contains an E-box in the P1 promoter; consequently, its expression can be regulated directly by BMAL1/NPAS2 and BMAL1/CLOCK heterodimers, which can be induced by Per2 (23). The p53 expression also exhibited daily oscillation at the mRNA and protein levels in the C57BL/6J mouse heart. However, no such rhythm was observed in the liver of 129/C57BL6 mice (23). This divergence may be due to tissue-specific expression of the gene.
Surprisingly, we found that c-Myc and p53 lost their circadian rhythms completely in both groups of ApoE−/− mice. The results suggest that other factors may play important roles in the regulation of the two genes in atherosclerosis because clock genes involved in their regulation retained daily oscillations despite the expression changes. The changes of these clock-controlled apoptosis-related genes are more significant than the clock genes in atherosclerosis. It seems that clock genes are more stable than apoptosis-related genes in the process.
Both c-Myc and p53 have important roles in atherosclerosis. c-Myc regulates cell growth and proliferation (24). c-Myc activation and target genes may have long-term effects on smooth muscle cell proliferation, which may contribute to the development of atherosclerotic lesions (25,26). The tumour suppressor protein p53, is involved in both cell proliferation and apoptosis, and is upregulated by various inducers of cellular stress known to be present in an atheromatous setting (27). p53 expression is increased in human atherosclerotic lesions, both in lipid-laden macrophages and smooth muscle cells (28). Vascular smooth muscle cells isolated from atherosclerotic plaques are more susceptible to p53-mediated apoptosis than normal vascular smooth muscle cells (29). There is also evidence that overexpression of p53 leads to plaque rupture by inducing apoptosis under certain experimental conditions (30). Thus, loss of p53 and c-Myc expression rhythms will lead to abnormal cell growth, proliferation and apoptosis in vivo that can affect the form, progress and even rupture of plaques in ApoE−/− mice.
We determined the profiles of 113 atherosclerosis-related genes by microarray to find their possible abnormalities in atherosclerosis. The results showed that some atherosclerosis-related genes have different expression levels at ZT0 and ZT12 in the two strains of mice. These genes may be regulated by the circadian clock and are classified as CCGs, and they exhibited such time-dependent behaviour. C57BL/6J mice had many more such time-dependent genes and most lost this trait in ApoE−/− atherosclerotic mice. Only a few time-dependent genes were shared by the two strains of mice, suggesting that the time-dependent manner of these genes were not affected in the process of atheroslerosis. Thus, we speculate that most clock-controlled atherosclerosis-related genes, such as p53 and c-Myc, were affected in atherosclerosis. These changes may play important roles in cardiovascular disease.
Our data demonstrated for the first time that clock genes, apoptosis- related genes and atherosclerosis-related genes exhibit altered expression levels and/or rhythms in the process of atherosclerosis in ApoE−/− mice. With the development of the disease, the disturbances of clock genes tended to become more severe and abnormalities of apoptosis-related genes were even more severe. The alterations in the expression of these genes may play important roles in the development of atherosclerosis, although the mechanisms behind these phenomena are not yet understood. Further studies in this field will provide new insights to better understand the role of the circadian clock in atherosclerosis.