Circadian disruption with constant light exposure exacerbates atherosclerosis in male ApolipoproteinE-deficient mice

Disruption of the circadian system caused by disordered exposure to light is pervasive in modern society and increases the risk of cardiovascular disease. The mechanisms by which this happens are largely unknown. ApolipoproteinE-deficient (ApoE−/−) mice are studied commonly to elucidate mechanisms of atherosclerosis. In this study, we determined the effects of light-induced circadian disruption on atherosclerosis in ApoE−/− mice. We first characterized circadian rhythms of behavior, light responsiveness, and molecular timekeeping in tissues from ApoE−/− mice that were indistinguishable from rhythms in ApoE+/+ mice. These data showed that ApoE−/− mice had no inherent circadian disruption and therefore were an appropriate model for our study. We next induced severe disruption of circadian rhythms by exposing ApoE−/− mice to constant light for 12 weeks. Constant light exposure exacerbated atherosclerosis in male, but not female, ApoE−/− mice. Male ApoE−/− mice exposed to constant light had increased serum cholesterol concentrations due to increased VLDL/LDL fractions. Taken together, these data suggest that ApoE−/− mice are an appropriate model for studying light-induced circadian disruption and that exacerbated dyslipidemia may mediate atherosclerotic lesion formation caused by constant light exposure.

(p = 0.68), and white adipose tissue (p = 0.66) did not differ significantly between 8-week old ApoE +/+ mice and ApoE −/− mice at 8 and 20 weeks old (one-way ANOVA; Fig. 3a, Table S2). Likewise, we found that the phases of the SCN (p = 0.54), liver (p = 0.87), pituitary (p = 0.36), lung (p = 0.15), kidney (p = 0.59), aorta (p = 0.62), spleen (p = 0.41), and white adipose tissue (p = 0.73) were not significantly different between ApoE +/+ and ApoE −/− mice at 8 and 20 weeks old (one-way ANOVA, Fig. 3b, Table S2). The amplitudes of the tissue PER2::LUC rhythms were also similar between 8-week old ApoE +/+ mice and ApoE −/− mice at 8 and 20 weeks old (Table S2). These data demonstrate that the molecular timekeeping mechanism is not altered by APOE deficiency. exposure to constant light exacerbates atherosclerosis in male ApoE −/− mice. We next investigated the effects of chronic exposure to constant light on atherosclerosis (Fig. 4, Table S3). Similar to previous studies in wild-type mice, locomotor activity was arrhythmic or the rhythm was disrupted in ApoE −/− mice housed in constant light (Fig. 4b, actograms of all individual mice shown in Fig. S10, S11, S12, S13; Fig. S14) 35 . Male (Fig. 4c, t-test p = 0.001), but not female (Fig. 4e, Mann-Whitney p = 0.08) ApoE −/− mice housed in constant light had more atherosclerosis in the en face aorta compared to those in control 12 L:12D (Table S3). Likewise, atherosclerotic lesion area in the aortic root was increased in male (Fig. 4d, Mann-Whitney p = 0.04), but not in female (Fig. 4f, t-test p = 0.20), ApoE −/− mice exposed to constant light (Table S3). Representative single-plotted actograms of wheel-running activity of ApoE +/+ (a) and ApoE −/− (b) mice housed in 12 L:12D for 7 days (LD) and then released into constant darkness for 7 days (DD). Yellow shading shows lights on. Mean activity profiles (c) were generated from 7 days in 12 L:12D. The amplitudes in LD (d) were the peak Qp's of the χ2 periodograms for 7 days in 12 L:12D. The phase angles of entrainment (e) were determined by drawing a regression line to activity onset for days 1-5 in constant darkness and then extending the line to the last day in 12 L:12D. A positive phase angle occurred when activity started after the time of lights off. The free-running period (f) was determined using an χ2 periodogram for days 1-7 in constant darkness. There were no significant differences between ApoE +/+ and ApoE −/− mice. Data are mean ± SEM. www.nature.com/scientificreports www.nature.com/scientificreports/ Constant light exposure exacerbates dyslipidemia, but not inflammation in male ApoE −/− mice. We next examined the potential mechanisms by which exposure to constant light could increase atherosclerosis in male ApoE −/− mice. Male ApoE −/− mice had similar body weights (t-test p = 0.13) and calorie consumption (t-test p = 0.52) in 12 L:12D and constant light, although the mice were less active in constant light (Mann-Whitney p = 0.008) (Table S4).
We next measured macrophages in the vascular wall of aortic roots as a marker of inflammation (Fig. 6). The percent macrophage content of the lesions in aortic roots was not altered by constant light exposure in male ApoE −/− mice compared to mice in 12 L:12D (Fig. 6c, t-test p = 0.62, Table S3).

Discussion
ApoE −/− mice are a well-established rodent model for studying atherosclerosis 36 . Beginning at ~12 weeks of age, male and female ApoE −/− mice spontaneously develop atherosclerotic lesions in the aorta, even when fed a low-fat (non-Western) diet 34 . It was critical to our experimental design to use a mouse model that develops atherosclerosis on low-fat diet. This is because diet-induced obesity increases atherosclerosis in ApoE −/− mice and high-fat diet feeding disrupts daily rhythms in male wild-type mice 37,38 . Therefore, in this study, we sought to exclude the potential confounding effects of high-fat diet feeding on atherosclerosis and circadian rhythms in order to isolate www.nature.com/scientificreports www.nature.com/scientificreports/ the effects of constant light-induced circadian disruption. To this end, we fed ApoE −/− mice a low-fat diet for the duration of the study. This protocol prevented hyperphagia and obesity in male ApoE −/− mice. Thus, the exacerbation of atherosclerosis in male ApoE −/− mice housed in constant light occurred independently of systemic metabolic dysfunction caused by obesity.
The first goal of this study was to determine if ApoE −/− mice are an appropriate model for studying the effects of light-induced circadian disruption on atherosclerosis. An ideal model should have no inherent circadian rhythm abnormalities. One previous study found that ApoE −/− mice had unstable activity rhythms in constant darkness and impaired circadian responsiveness to light 39 . Therefore, we first comprehensively characterized circadian rhythms of behavior and light responsiveness, and molecular circadian rhythms in tissues in ApoE −/− mice. We found that ApoE −/− mice were indistinguishable from ApoE +/+ mice in every parameter measured, with the exception of slight significant reductions in total activity and amplitude in ApoE −/− mice in constant darkness. Endogenous (free-running) and entrained rhythms of wheel-running activity, as well as responsiveness to light pulses at different times of day were the same in ApoE −/− and ApoE +/+ mice. Moreover, the molecular timekeeping rhythms in the SCN and peripheral tissues, as well as the phase relationship between these body clocks, were the same in ApoE −/− and ApoE +/+ mice. We postulate that our results differ from the previous study because the ApoE −/− mice used in the prior study may have been on a mixed genetic background while the ApoE −/− mice in our study were backcrossed 10 times into a C57BL/6 J background. Genetic background markedly affects www.nature.com/scientificreports www.nature.com/scientificreports/ circadian activity rhythms in mice and the C57BL/6 J strain has stable, high-amplitude behavior rhythms 40 . In sum, we found that ApoE −/− mice have no circadian abnormalities and thus are an ideal model for studying the effects of light-induced circadian disruption on atherosclerosis.
The second goal of this study was to investigate the effects of constant light exposure, which chronically disrupts circadian rhythms, on atherosclerosis. Most previous studies investigated the effects of circadian disruption on atherosclerosis using circadian gene mutant mice [15][16][17][18][19] . To our knowledge, only two studies have examined the effects of light-induced circadian disruption on atherosclerosis in mice. One study inferred that a weekly inversion of the light-dark cycle accelerated atherosclerosis development in ApoE −/− mice, but lacked quantitative data 41 . A second study recently showed that weekly inversion of the light-dark cycle increased severe atherosclerotic lesions www.nature.com/scientificreports www.nature.com/scientificreports/ in female APOE*3-Leiden.CETP mice due to increased macrophage content and inflammation, oxidative stress, and chemoattraction markers in the vascular walls 42 . However, the data from the present study demonstrate that the increase in atherosclerosis in male ApoE −/− mice exposed to constant light was driven by increased VLDL/ LDL cholesterol rather than increased macrophage content. Additionally, the mice in the previous study were fed a Western diet (high fat and high cholesterol), which independently causes metabolic dysfunction, obesity, and disrupts circadian rhythms 27,[43][44][45][46][47][48] . Therefore, our study is unique in that we found that disordered light exposure exacerbates atherosclerosis even when mice are fed low-fat diet. An additional strength of our study is that we studied both male and female ApoE −/− mice, while only females could be studied in the previous study because male APOE*3 Leiden.CETP mice do not develop atherosclerosis on a cholesterol-rich diet.
In mice, constant light exposure increases the period of activity rhythms and, chronically, can cause arrhythmicity 49 . At the tissue level, constant light desynchronizes cellular oscillators in the SCN, causing the overall rhythm of the SCN to be low-amplitude or absent 35 . Since the SCN is the master circadian clock that coordinates physiological and behavioral rhythms throughout the body, the effect of constant light exposure on the SCN results in whole-body disruption of circadian rhythms 35,50 . Previous studies showed that constant light exposure increased body weight, disrupted insulin sensitivity, and decreased triglyceride-derived fatty acids and glucose uptake by brown adipose tissue [51][52][53] . However, no study has examined the effects of constant light exposure on atherosclerosis. In the current study, we found that exposure to constant light increased cholesterol on atherogenic lipoproteins and atherosclerotic lesion area in male ApoE −/− mice. According to the lipid hypothesis, chronic elevated levels of cholesterol in the blood causes atherosclerosis [54][55][56] . In female ApoE −/− mice, constant light exposure did not increase total or VLDL/LDL-cholesterol concentrations nor atherosclerotic lesion area. The mechanisms underlying the sex difference in response of lipids to light-induced circadian disruption are unknown but could be due to differences in circulating sex hormones.
In sum, we found that ApoE −/− mice had circadian rhythms that were indistinguishable from ApoE +/+ mice. In addition, circadian disruption with constant light exposure increased atherosclerosis in male, but not female ApoE −/− mice. Together these data establish male ApoE −/− mice as an appropriate model for studying the effects of light-induced disruption of circadian rhythms on atherosclerosis.  www.nature.com/scientificreports www.nature.com/scientificreports/ Methods Animals. C57BL/6 J ApoE −/− (N10) mice were purchased from The Jackson Laboratory (stock # 002052) and bred with wild-type C57BL/6 J mice (from The Jackson Laboratory) to generate C57BL/6 J heterozygous ApoE +/mice. Heterozygous C57BL/6 J ApoE +/males and females were bred to generate ApoE +/+ and ApoE −/− mice (N11-N12) for experiments. For bioluminescence experiments, ApoE +/mice that were heterozygous for PERIOD2::LUCIFERASE 33 (originally obtained from Dr. Joseph Takahashi and then backcrossed for 25 generations with C57BL/6 J mice from The Jackson Laboratory) were crossed with ApoE +/mice to generate ApoE +/+ and ApoE −/− mice that were heterozygous for PER2::LUC for experiments. Breeders and weanlings were housed in 12 L:12D and fed standard rodent laboratory diet (Teklad 2918) and water ad libitum. At 3 weeks old, offspring were weaned and group-housed with same-sex siblings. Genotyping for ApoE −/− was performed according to the protocol on The Jackson Laboratory website. Genotyping for PER2::LUC was determined by measuring bioluminescence from tail snips. For all experiments, mice were singly-housed in cages (33 cm × 17 cm × 14 cm) with running wheels (diameter: 11 cm) and fed low-fat diet (Research Diets D12450K, 10% kcal fat) and water ad libitum. The running wheels were either unlocked (could rotate) or locked (could not rotate), as indicated for each specific experiment below. All procedures were conducted in accordance with animal protocol 2015-2211 approved by the University of Kentucky Institutional Animal Care and Use Committee.
Characterization of circadian behavior. At 7 to 8 weeks old, male and female ApoE −/− and ApoE +/+ mice were housed singly in cages with unlocked running wheels. The cages were placed in light-tight boxes in 12 L:12D with white LED lights (intensity 250 to 350 lux). Wheel revolutions were recorded every minute using the ClockLab system (Actimetrics, Inc, Wilmette, IL). Mice were housed in 12 L:12D for 7 days and then in constant darkness for 7 days. Data were analyzed with ClockLab analysis software (Actimetrics). Mean activity profiles of wheel-running activity (5-min bins) were compiled for7 days in 12 L:12D. The amplitude (Q p ) of the wheel-running rhythm was the peak value of the χ 2 periodogram for 7 days in 12 L:12D or 7 days in constant darkness or 7 days in constant light. Mean daily activity was determined for 7 days in 12 L:12D or 7 days in constant darkness or 7 days in constant light. The phase angle of entrainment was determined by fitting a linear regression line to 5 days in constant darkness, and then extending it back to the last day in 12 L:12D. The free-running period of wheel-running activity in constant darkness was determined by χ 2 periodogram with alpha set to 0.001 for days 1-7 in constant darkness. After 18 days in constant darkness with weekly light pulses (see below), we returned the mice to 12 L:12D for 20 days and then released them into constant light for 21 days. The free-running period of wheel-running activity in constant light was determined by χ 2 periodogram for days 1-7 in constant light. Wheel-running activity data are shown in actograms in 6-min bins in the normalized format (ClockLab).
Circadian phase responses to light pulses. Male and female ApoE +/+ and ApoE −/− mice (12-18 weeks old at time of light pulse) were single housed in cages with ad libitum access to unlocked running wheels. The cages were housed in light-tight boxes in 12 L:12D with white LED lights (intensity 250 to 350 lux). Mice were then released into constant darkness for 7 days. The onset of activity on the day of the light pulse, which was designated as CT12, was determined by linear regression using ClockLab Analysis. A single light pulse (15 min, 150 lux white LED) was administered at CT8-10 (subjective day), or CT14-16 (early subjective night), or CT21-23 (late subjective night). The mice then free-ran for 7 days after the light pulse. The magnitudes of the phase shifts were determined by measuring the time between a line fit to the onset of activity the 7 days before the light pulse and a line fit to the onset of activity the 7 days after the light pulse using ClockLab Analysis software. Some mice were administered more than 1 light pulse, every 3 weeks, and the cages were changed in the week between pulses. Each mouse received no more than 3 light pulses and an individual mouse never received a light pulse at the same CT.
Bioluminescence tissue rhythms. Male and female ApoE −/− and ApoE +/+ mice (7 weeks old) that were heterozygous for PER2::LUC were housed singly in cages with locked wheels (wheels were present but could not rotate) in 12 L:12D. At 8 or 20 weeks old, mice were euthanized by cervical dislocation without anesthesia and aorta, kidney, lung, liver, pituitary, spleen, SCN, and white adipose tissue explants were dissected and cultured as described previously 57 . Bioluminescence was measured every 10 min with the 32-channel LumiCycle apparatus (Actimetrics Inc.). Data were smoothed by 30-min adjacent averaging and detrended using LumiCycle Analysis software (Actimetrics Inc.). The amplitude (goodness of fit ≥90%) was determined from the cycle that occurred between 12-36 hours in culture with LumiCycle Analysis software (Actimetrics). The data were exported to ClockLab analysis software to analyze period and phase. The period was determined from a regression line fit to the acrophase of 3-5 cycles. The phase was the acrophase of the peak of bioluminescence that occurred between 12-36 hours in culture.
Effects of constant light exposure on atherosclerosis, lipids, and inflammation. At 7 weeks old, male and female ApoE −/− mice were single-housed in cages with locked running wheels in light-tight boxes in 12 L:12D. General locomotor activity was continuously recorded with passive infrared sensors and collected in one-minute intervals using the ClockLab acquisition system (Actimetrics Inc.). At 8 weeks old, mice were randomized to either remain in 12 L:12D or to be housed in constant light for 12 weeks. Body and food weights were measured weekly between ZT9-ZT12, where ZT0 is lights on and ZT12 is lights off, in 12 L:12D, or the corresponding local time for mice in constant light. χ 2 periodograms were used to determine whether general locomotor activity was rhythmic in the final 28 days in constant light using ClockLab Analysis software. Activity was rhythmic if a dominant peak exceeded alpha set at p = 0.001 (period range 20-36 h).

Scientific RepoRtS |
(2020) 10:9920 | https://doi.org/10.1038/s41598-020-66834-9 www.nature.com/scientificreports www.nature.com/scientificreports/ At 20 weeks of age, mice were anesthetized with inhaled isoflurane between ZT6-9 (or the corresponding local time for mice in constant light) until unresponsive to toe pinches, then euthanized by cervical dislocation. Blood was collected by cardiac puncture and serum was separated by centrifugation and stored at −80 °C. The aortas were perfused with NaCl (0.9% wt/vol) via left ventricular puncture. Aortas were dissected from the root to the iliac bifurcation as described previously 58 . The heart was removed from the aorta and stored at −80 °C. Mice with gross organ abnormalities were excluded from the study (2 mice from the female constant light group were excluded for enlarged kidneys). The aortas were stored in 10% neutrally-buffered formalin for 24-hours at room temperature and then transferred to 0.9% NaCl solution and stored at room temperature.
To measure atherosclerotic lesion area in en face aortas, peri-aorta adipose tissue was removed and the aorta was cut longitudinally and photographed using Image-Pro 7.0 software. The aortic arch was defined as the ascending arch to 3 mm distal to the root of the left subclavian artery. Atherosclerotic lesion area of the aortic arch was measured using Image-Pro 7.0 software by one researcher not blinded to the experimental treatments and analyzed by a second researcher who was blinded to the experimental groups.
Atherosclerotic lesion area in the aortic root was measured as described previously 58 . Briefly, 9 serial tissue sections (10 µm) from the origin of the aortic valves to the ascending aorta, were stained with oil red O. Serial sections were distributed among eight consecutive slides resulting in ~80 µm intervals for each slide. Lesions were quantified from the internal elastic lamina to the luminal edge using Image-Pro 7.0 software by 2 researchers as described above. Macrophage quantification. Immunohistochemistry for macrophages was performed on frozen serial sections (adjacent to those stained with Oil Red O) of the ascending aorta using the MicroProbe system as described previously using rat anti-mouse CD68 (Bio-Rad, Cat# MCA1957), Rat IgG2b (BD PharMingen, Cat# 559478), and biotinylated rabbit anti-rat IgG (Vector, Cat# BA-4001) antibodies 59 . Immunoreactivity was visualized with red chromogen 3-amino-9-ethylcarbazole (AEC) (Vector). Macrophage content was quantified in the first serial section of the aortic root from the internal elastic lamina to the luminal edge using Image-Pro 7.0 software by 2 researchers as described above. The area of CD68 staining was expressed as a percentage of atherosclerotic lesion area.

Statistical analyses.
A priori power analyses were performed for circadian behavior parameters and en face atherosclerotic lesion area using alpha = 0.05, power = 0.80, and effect size of 1 to 1.5 using G*Power (Heinrich Heine Universität Düsseldorf) (Table S5). Circadian behavior parameters (amplitude, daily activity, phase angle of entrainment, period) and phase shifts in reponse to light pulses at each CT were compared between ApoE +/+ and ApoE −/− mice using two-tailed Student's t-tests, unless the data were not normally distributed or had unequal variance, in which case the Mann-Whitney test was used. One-way ANOVAs were used to compare the periods, phases, and amplitudes of the bioluminescence rhythms of each tissue among groups. Atherosclerosis lesion area in the en face aorta and aortic roots, total serum cholesterol and triglyceride concentrations, cumulative food intake, cumulative locomotor activity, and the percentage of lesions with macrophages in ApoE −/− mice of the same sex were compared between 12 L:12D and constant light conditions using two-tailed Student's t-tests, unless the data were not normally distributed or had unequal variance, in which case the Mann-Whitney test was used. Statistical tests were performed with OriginPro 2016 (Northampton, MA). Data are presented as the mean ± SEM. Significance was ascribed at p < 0.05.

Data availability
All data generated or analyzed during this study are included in this published article and its Supplementary Information files.