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Expression Changes of Circadian Genes in SCN, Heart and Liver in Apolipoprotein E Knock-out Atherosclerotic Mice

Author: HouLiKun
Tutor: QianZuoZhe;JinHuiMing;ChenSiFeng;LuChao
School: Fudan University
Course: Pathology and Pathophysiology
Keywords: Circadian clock genes Atherosclerosis Lipid metabolism disorders apoE gene knockout mice Transcription factor
CLC: R543
Type: Master's thesis
Year: 2009
Downloads: 48
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Abstract

The circadian clock is involved in regulating behavior and many physiological functions. Circadian rhythm’ generation is based on the coordination expression of the core circadian genes through the transcription-translation feedback loops. The master circadian clock is located in suprachiasmatic nucleus (SCN) of the hypothalamus, which is thought to drive peripheral oscillators by controlling neuronal and humoral signals that can entrain the peripheral clocks. By regulating the downstream clock controlled genes’ (CCGs) expression, circadian clock takes part in many physical functions. Many researches have showed that change of circadian rhythmicity plays an important role in several pathological processes such as cardiovascular and neurologic diseases, tumor.In cardiovascular disease, the occurrence of stroke, acute myocardial infarction and sudden cardic death showed a circadian rhythm with the peak in the morning hours. Although the precise mechanism underlying the phenomenon is still not clear, they are mainly characterized by atherosclerosis, which is a chronic vascular disease resulted by complicated causes including inflammation, disorder of lipid metabolism and hemostasis. In the physiological process, circadian clock takes part in lipid metabolism by clock controlled genes related lipid metabolic enzyme. Dysfunction of this pathway resulted in metabolic syndrome including dyslipidemia, insulin resistance, obesity, hypertension. High plasma concentrations of cholesterol resulted in endothelial cell activation and dysfunction causing the release of vasoactive molecules and cytokines, increased expression of adhesion molecules within the atherosclerotic lesion stimulates monocyte recruitment and transmigration into the arterial intima and accumulation of lipids. Intracellular accumulation of cholesterol results in the characteristic formation of foam cells, enhanced cytokine release causes progressive enlargement of the plaque. The plaque ruptures resulted in thrombus formation and tissue infarction. Whether disorder of lipid metabolism as a risk factor in the process of atherosclerosis can affect the clock genes’ expression? Our previous research demonstrated the expression of clock genes and CCGs was changed in hearts of atherosclerotic mice resulted by disorder of lipid metabolism, whereas, the expression of clock genes was affected by a high-fat diet and took part in regulating the lipid metabolism. Therefore, we predict that expression changes of clock genes was related with disorder of lipid metabolism, rhythmic disorder affected the lipid metabolism, resulting in infernal cycle of lipid metabolism, further playing an important role in the pathological process atherosclerosis. In present study, apoE knock-out (apoE-/-) mice fed regular chow or a hight-fat diet were used to establish the atherosclerotic animal model of early stage and advanced stage. We identified whether and how circadian rhythmic expression of clock genes was changed in SCN involved in regulation of many pathophysiological functions and heart playing a role in the process of atherosclerosis and liver related to the lipid metabolism, to illustrate the relationship between disorder of lipid metabolism and expression changes of clock genes in the process of atherosclerosis.36 male apoE-/- mice and 18 male C57BL/6J control mice were kept in 12-hour light/12-hour dark contidion for 2 weeks, then transferred to 12-hour dark/12-hour dark contidion for another 3 weeks. apoE-/- mice were divided into two groups: one group (n=18) were fed regular chow, the other were fed a high-fat diet (containing 0.15% cholesterol and 21% fat). According to circadian time (CT circadian time used for assessing biological time without any time cues; CT0 designates the beginning of the subjective dawn and CT12 that of the subjective dusk), mice were sacrificed at different time points (n=3 per time point), namely, CT0, CT4, CT8, CT12, CT16 and CT20. The serum was prepared for total cholesterol, LDL-CHO and HDL-CHO detection. The aorta roots were prepared for frozen sections. Oil Red O staining method was used to detect foam cells in subendothelium and atherosclerotic plagues. The rhythmic expression of clock genes (mPer2, mBmal1, mClock, mCry1 and mRev-erbα) was detected by Real-time PCR. The results suggested that no obvious plagues were found in apoE-/- mice fed regular chow, foam cells were observed under the endothelium. The serum concentration of cholesterol and LDL-CHO was increased and HDL-CHO concentration was decreased. After feeding a high-fat diet for 5 weeks, atherosclerotic lesions formed in apoE-/- mouse aortas. The cholesterol and LDL-CHO levels were elevated more highly in the serum, and the HDL-CHO concentration was decreased more severely. Thus, apoE-/- mice fed regular chow and a high-fat diet were at the early stage and advanced stage of atherosclerosis, respectively.Real-time PCR results showed that circadian clock genes in the central and peripheral tissues of apoE-/- and C57BL/6J mice exhibited daily oscillations except mClock in SCN and heart. In SCN, the amplitude of clock genes’ expression wasn’t changed in apoE-/- mice, but the rhythmicity of clock genes was different. In heart, the expression of mPer2 mRNA was lower at CT0, CT4 and higher at CT12, CT16 in apoE-/-mice than that of C57BL/6J mice. In contrast, the amplitude of mBmal1 mRNA expression was elevated at CT0 and decreased at CT12 and CT16 in apoE-/-mice. The peak and the trough of mBmal1 mRNA level delayed four hours and occurred at CT0 and CT12, compared to C57BL/6J mice. At CT12, CT16 and CT20, the amplitude expression of mCry1 mRNA was higher in apoE-/-mice fed a high-fat diet than that in apoE-/-mice fed regular chow. In the apoE-/- mice livers, the expression of clock genes mBmal1, mPer2, mCry1 and mClock mRNA was decreased at several time points, compared to that of C57BJ/6L mice.The amplitude and rhythmicity of mRev-erbαmRNA was affected only during the light period in both central and peripheral tissure in apoE-/-mice in comparison with C57BL/6J mice. Compared to the peak at CT0 in C57BL/6J mice, the peak of mRev-erbαmRNA was four hours delayed and reached at CT8, CT12 in apoE-/- mice fed regular chow or a high-fat diet, respectively.Above results showed the expression amplitude and rhythmicity of all the target clock genes was changed in apoE-/-mice. The similar expression changes of clock genes may be regulated by the individual or several transcription factors in their upstream response region, in which hundreds of transcription factors were analyzed, of these, transcription factors such as mRev-erbα, mPPARα, mRXRα, mHNF-3α, mHNF-3β, mPPARγthat had high frequent binding sites in clock genes’ upstream response region. Among the mutual transcription factors, mRev-erbα, mPPARαand mRXRαrelated with lipid metabolism and cardiovascular diseases were detected in SCN, hearts and livers of C57BL/6J and apoE-/-mce. The results showed the expression pattern of mPPARαand mRXRαmRNA in SCN and hearts of C57BL/6J and apoE-/-mice wasn’t changed, whereas, in livers, expression of transcription factors’ mPPARαand mRXRαwere observed daily oscilliation. But the amplitude of mPPARαand mRXRαmRNA expression of apoE-/-mice fed a hight-fat diet was increased at CT0, CT4 and CT8, compared to that of apoE-/-mice fed regular chow.In summary, apoE-/-mice were used to establish the atherosclerotic animal model. The rhythmicity of circadian genes was affected a little in SCN. In apoE-/-mice hearts, the peak and the trough of circadian genes mRNA were delayed and accompanied with expression changes of amplitude, compared to C57BL/6J mice. But in livers of apoE-/-mice fed a high-fat diet, the amplitude of all the target clock genes’ expression was decreased at several time points, meanwhile, expression of transcription factors’ mPPARαand mRXRαmRNA was increased in apoE-/-fed a high-fat diet, which demonstrated that lipid metabolism related genes’ mPPARαand mRXRαexpression was negative correlation with circadian genes’s expression in livers. Disorder lipid metabolism affected the expression and rhythmicity of clock genes, the mechanism may be related with the change expression of transcription factor of the clock genes. Different expression change of clock genes was shown in different tissures. These changes may be play an inportant role in the pathological process of atherosclerosis and the precise role and the mechanisms are to be elucidated.

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