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Anea et al. Vessel Plus 2018;2:16 I http://dx.doi.org/10.20517/2574-1209.2018.46 Page 3 of 8

Immunohistochemistry
Vascular tissue samples were dissected from regular wild type C57Bl6 mice, and rapidly embedded for
frozen cross-sectioning. Sections were cut at 5 μm and mounted onto glass slides. Afterwards, Clock
components were immunohistochemically detected. Briefly, the indirect avidin biotin-horseradish peroxidase
visualization method was used (ABC Standard and Elite, Vector Red, Vector Laboratories, Burlingame,
USA). Samples were incubated with the detection primary antibody at the manufacturer’s recommended
concentration.

RESULTS
To determine the cellular and temporal expression of the vascular clock, we harvested tissues from wild-
type mice at 12 AM and 12 PM and conducted immunohistochemical analysis of different circadian clock
components. At 12 AM, the common carotid artery, aorta, and femoral artery and vein exhibited Bmal1
expression that was largely delineated in the outer adventitial region of the blood vessel [Figure 1A].
Smooth muscle cell expression and endothelial expression were virtually absent. Similarly, at 12 AM, heart
and lung exhibited low expression of Bmal1. At 12:00 PM, adventitial Bmal1 expression was reduced, while
smooth muscle and endothelial Bmal1 was increased in carotid, aorta, and femoral artery and vein. In
heart, there was increased Bmal1 staining that was also evident in lung. Clock staining exhibited enhanced
endothelial positivity at 12 AM and was virtually absent adventitial staining in contrast to Bmal1, but
did exhibit increased overall tissue expression at 12 PM similar to Bmal1 [Figure 1B]. Cry 1 exhibited
little adventitial staining in carotid arteries at either time point, but medial (smooth muscle) staining was
increased at 12 PM, as it was also observed in aorta [Figure 1C]. In femoral artery and vein, in contrast
to Bmal1 and Clock, Cry exhibited robust expression at 12 PM in the adventitia. In both heart and lung,
Cry1 positive cells were robustly increased at 12 PM, with a punctate nuclear stain. In carotid artery and
aorta at 12 AM, casein kinase expression was robust, but restricted to the adventitia, and became diffuse
in the media at 12 PM [Figure 1D]. Femoral artery, vein, heart and lung followed expression patterns that
aligned with the other circadian clock components. Npas2 exhibited a distinctive adventitial staining in
all three vessel beds examined, different from the other clock components that occurred at 12 PM, and
exhibited punctate nuclear staining in heart tissue and more diffuse staining in lung [Figure 1E]. Ror
exhibited medial staining in the blood vessels but most distinctive was in lung tissue, where epithelial cells
of bronchioles were highly positive, unique from other clock components [Figure 1F]. Per1 exhibited a
strong medial expression in all vascular tissues [Figure 1G, first 3 panels], and also a diffuse expression in
heart and lung [Figure 1G, last 2 panels].

To complement the study, we next assessed circadian clock expression in the human saphenous vein [Figure 2].
Bmal1 and Clock expression were increased throughout the media at 12 PM relative to 12 AM and exhibited
strong endothelium staining. Npas2 staining was relatively absent in the saphenous vein, while Per1 exhibited
increased expression at 12 AM relative to 12 PM. Cry1 did not exhibit any temporal difference in expression
but was expressed throughout the media. Rev-erbα expression was distributed throughout the media and
did not exhibit a temporal expression pattern while Rora and Epas were increased in the media at 12 PM.
Casein kinase was not robustly expressed in the saphenous but was increased at 12 PM. We then examined
saphenous vein expression of the positive limb component Bmal1 and negative limb component Cry and
examined expression by western blot [Figure 3]. Bmal1 trough occurred at 6 PM, while Cry peaked at 6 PM,
and Bmal1 peaked at 15:00 (3 PM) while Cry1 was at nadir at 18:00, consistent with the antiphase nature of
the positive and negative limb components.

DISCUSSION
The circadian clock is a cell synchronized signaling pathway that serves to control timing. Within the
cardiovascular system, heart [17,18] , vascular [2,19,20] , lung [21,22] , and even kidney circadian clocks [23,24] are