Genetic components of the circadian clock regulate thrombogenesis in vivo.

BACKGROUND: Myocardial infarction, stroke, and sudden death undergo diurnal variation. Although genes relevant to hemostasis and vascular integrity undergo circadian oscillation, the role of the molecular clock in thrombotic events remains to be established. METHODS AND RESULTS: A diurnal variation in the time to thrombotic vascular occlusion (TTVO) subsequent to a photochemical injury was observed in wild-type mice: TTVO varied from 24.6+/-2.7 minutes at zeitgeber time (ZT) 2 to 40.3+/-4.3 minutes at ZT8, 24.3+/-2.3 minutes at ZT14, and 31.0+/-4.4 minutes at ZT20. This pattern was disrupted or altered when core clock genes-BMAL1, CLOCK, and NPAS2-were mutated or deleted. Mutation of CLOCK abolished the diurnal variation in TTVO, whereas deletion of NPAS2 altered its temporal pattern. NPAS2 deletion prolonged TTVO and reduced blood pressure irrespective of clock time. Global BMAL1 deletion shortened TTVO at ZT8, and the diurnal variation in TTVO, but not in systemic blood pressure, was disrupted in mice in which BMAL1 had been selectively deleted in endothelium. CONCLUSIONS: Key components of the molecular clock regulate the response to a thrombogenic stimulus in vivo. Such a phenomenon may interact with environmental variables, and together with the influence of these genes on blood pressure may contribute to the diurnal variation in cardiovascular events observed in humans.

Peripheral circadian clock rhythmicity is retained in the absence of adrenergic signaling.

OBJECTIVE: The incidence of heart attack and stroke undergo diurnal variation. Molecular clocks have been described in the heart and the vasculature; however it is largely unknown how the suprachiasmatic nucleus (SCN) entrains these peripheral oscillators. METHODS AND RESULTS: Norepinephrine and epinephrine, added to aortic smooth muscle cells (ASMCs) in vitro, altered Per1, E4bp4, and dbp expression and altered the observed oscillations in clock gene expression. However, oscillations of Per1, E4bp4, dbp, and Per2 were preserved ex vivo in the aorta, heart, and liver harvested from dopamine beta-hydroxylase knockout mice (Dbh-/-) that cannot synthesize either norepinephrine or epinephrine. Furthermore, clock gene oscillations in heart, liver, and white adipose tissue phase shifted identically in Dbh-/- mice and in Dbh+/- controls in response to daytime restriction of feeding. Oscillation of clock genes was similarly preserved ex vivo in tissues from Dbh+/- and Dbh-/- chronically treated with both propranolol and terazosin, thus excluding compensation by dopamine in Dbh-/- mice. CONCLUSIONS: Although adrenergic signaling can influence circadian timing in vitro, peripheral circadian rhythmicity is retained despite its ablation in vivo.

Peripheral circadian clocks in the vasculature.

Living organisms have adapted to the daily rotation of the earth and regular changes in the light environment. Life forms anticipate environmental transitions, adapt their own physiology, and perform activities at behaviorally advantageous times during the day. This is achieved by means of endogenous circadian clocks that can be synchronized to the daily changes in external cues, most notably light and temperature. For many years it was thought that neurons of the suprachiasmatic nucleus (SCN) uniquely controlled circadian rhythmicity of peripheral tissues via neural and humoral signals. The cloning and characterization of mammalian clock genes revealed that they are expressed in a circadian manner throughout the body. It is now accepted that peripheral cells, including those of the cardiovascular system, contain a circadian clock similar to that in the SCN. Many aspects of cardiovascular physiology are subject to diurnal variation, and serious adverse cardiovascular events including myocardial infarction, sudden cardiac death, and stroke occur with a frequency conditioned by time of day. This has raised the possibility that biological responses under the control of the molecular clock might interact with environmental cues to influence the phenotype of human cardiovascular disease.

Histone acetyltransferase-dependent chromatin remodeling and the vascular clock.

Rhythmic gene expression is central to the circadian control of physiology in mammals. Transcriptional activation of Per and Cry genes by heterodimeric bHLH-PAS proteins is a key event in the feedback loop that drives rhythmicity; however, the mechanism is not clearly understood. Here we show the transcriptional coactivators and histone acetyltransferases, p300/CBP, PCAF, and ACTR associate with the bHLH-PAS proteins, CLOCK and NPAS2, to regulate positively clock gene expression. Furthermore, Cry2 mediated repression of NPAS2:BMAL1 is overcome by overexpression of p300 in transactivation assays. Accordingly, p300 exhibits a circadian time-dependent association with NPAS2 in the vasculature, which precedes peak expression of target genes. In addition, a rhythm in core histone H3 acetylation on the mPer1 promoter in vivo correlates with the cyclical expression of their mRNAs. Temporal coactivator recruitment and HAT-dependent chromatin remodeling on the promoter of clock controlled genes in the vasculature permits the mammalian clock to orchestrate circadian gene expression.