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. 2009 Mar 24;119(11):1510-7.
doi: 10.1161/CIRCULATIONAHA.108.827477. Epub 2009 Mar 9.

Vascular disease in mice with a dysfunctional circadian clock

Ciprian B Anea  1 , Maoxiang Zhang, David W Stepp, G Bryan Simkins, Guy Reed, David J Fulton, R Daniel Rudic
Affiliations

Vascular disease in mice with a dysfunctional circadian clock

Ciprian B Anea et al. Circulation. .

Abstract

Background: Cardiovascular disease is the leading cause of death for both men and women in the United States and the world. A profound pattern exists in the time of day at which the death occurs; it is in the morning, when the endothelium is most vulnerable and blood pressure surges, that stroke and heart attack most frequently happen. Although the molecular components of circadian rhythms rhythmically oscillate in blood vessels, evidence of a direct function for the "circadian clock" in the progression to vascular disease is lacking.

Methods and results: In the present study, we found increased pathological remodeling and vascular injury in mice with aberrant circadian rhythms, Bmal1-knockout and Clock mutant. In addition, naive aortas from Bmal1-knockout and Clock mutant mice exhibit endothelial dysfunction. Akt and subsequent nitric oxide signaling, a pathway critical to vascular function, was significantly attenuated in arteries from Bmal1-knockout mice.

Conclusions: Our data reveal a new role for the circadian clock during chronic vascular responses that may be of significance in the progression of vascular disease.

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Figures

Figure 1
Figure 1. Impaired remodeling and thrombosis in Bmal1-KO mice
(A) Morphometric analysis of common carotid artery cross sections revealed typical vascular remodeling in male, wild-type (WT) mice after 5 weeks of arterial ligation, evident as wall thickening of the medial layer while thickening of the LC wall was exacerbated in age-matched Bmal1-KO mice (young). (B) Masson-trichrome staining showed a robust deposition of collagen staining (blue stain) in the media of Bmal1-KO mice relative to wild-type mice (bar=50 μm). (C) In contrast to a reduction in lumen diameter observed in WT mice, lumen diameter of male Bmal1-KO mice did not narrow (young, 7-10 weeks) or a even exhibited a paradoxical increase diameter (old, 25-30 weeks) (D) in regions that were free of thrombosis. (E) Hematoxylin and eosin staining of remodelled LC of old wild-type (left panel) reveals a normal, inward remodelled vessel with patent lumen, while Bmal1-KO (right panel) exhibits remodeling an enlarged lumen undergoing remodeling around the point of fibrosis (bar=50 μm). (N=9-10, *p<0.05, paired t-test RC versus LC; †p<0.05, two-sample t-test LC-WT versus LC-KO) (F) Thrombosis was increased in old Bmal1-KO mice (N=7-10/age group, (*p<0.05, WT versus KO).
Figure 2
Figure 2. Clock mutant mice exhibit pathological vascular responses
(A) After acclimation to either light/dark (LD) or constant dark conditions (DD), the left common carotid artery of wild-type (WT) and Clock mutant (MUT) mice (male, 15 to 20 weeks of age) was ligated for 5 weeks and remodelled LC thickness was measured, revealing a DD-dependent increase in wall thickness of LC in Clockmut mice (N=4, *p<0.05, Clockmut in DD versus WT in DD) and a DD-dependent impairment in inward remodeling (B). (C) Von Gieson staining of elastin fibers revealed enhanced intimal hyperplasia in the left femoral artery of male, Clockmut mice undergoing intraluminal wire injury reveals an enhanced injury relative to injured arteries of male, WT mice, that were housed in DD (bar=50 μm). (D). Morphometric analysis of femoral artery cross sections revealed an increase in neointimal area and intima to medial ratio indicative of an exacerbated response to wire injury. (N=7-10, *p<0.05).
Figure 3
Figure 3. Activation of platelets is normal in Bmal1-KO mice, but endothelial and liver PAI-1 is increased
Platelet function was assessed in Bmal1-KO mice versus littermate control wild-type mice (male, 20-25 week old). Whole blood was isolated from mice and activated with PAR-4 to induce platelet aggregation, and analyzed for platelet markers by FACS analysis. There was no difference in total platelets (cd41) (A) or platelet activation (cd62) (B) in Bmal1-KO mice versus wild-type mice. (N=5, platelet aggregation studies *p<0.05, activated versus basal). (C) Protein expression of PAI-1 was increased in the endothelium (bar=30 μm) and livers (D) of 20-25 week old Bmal1-KO mice (liver immunoblot shows 3 wild type and 3 Bmal1-KO mice and right panel shows respective densitometry).
Figure 4
Figure 4. Endothelial dysfunction in mice with aberrant circadian rhythm
Isometric tension development was assessed in aortic rings prepared from Bmal1-KO mice (black squares) and litter-mate controls (white circles). After preconstriction with phenylephrine, concentration response to acetylcholine was measured. (A) Representative traces and summary data (B) of the acetylcholine response from wild-type and Bmal1-KO mice (10-15 week old, male) are shown. (C) Clock mutant mice were also assessed for their response to acetylcholine in dark-dark and light-dark conditions (D) (N=4 WT, N=6 Clockmut, *p<0.05 versus WT at corresponding concentration). (E) Relaxant responses to the endothelium-independent vasodilator SNP were not different between control and Bmal1-KO mice. (F) Ach response was assessed in WT and Bmal1-KO mice at 8 AM and 8 PM (N=5-7/group; *p<0.05, WT versus Bmal1-KO at respective time,†p<0.05 respective genotype at 8 AM versus 8PM)
Figure 5
Figure 5. Blunted Akt signaling in common carotid arteries of Bmal1 KO mice
(A) After 5 weeks of complete carotid artery ligation, remodelled arteries (LC) were dissected and protein lysates were isolated and separated by SDS-PAGE electrophoresis from littermate control wild-type mice versus Bmal1-KO mice at a single time point (1 PM). Expression levels of total Akt, P-Akt1, and the phosphorylating kinase PDK were dramatically reduced in remodelled left common carotid arteries relative to wild-type mice. Remodelled arteries of Bmal1-KO mice also exhibited blunted phosphorylated levels of phosphorylated eNOS, though total eNOS on average did not change. (B) Changes were quantified by densitometry (*p<0.05 versus WT). (N=4 Wild-type, N=4 Bmal1-KO). (C) After 1 week of LC ligation, proteins were assessed in both RC and LC, revealing a attenuated Akt and P-Akt expression in the unligated RC of Bmal1-KO mice relative to WT mice.
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