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Page 6 of 8 Anea et al. Vessel Plus 2018;2:16 I http://dx.doi.org/10.20517/2574-1209.2018.46

functional and rhythmic. Because organs are comprised of a heterogeneous population of cells, there are
likely cell-specific circadian profiles and expression patterns distinct to the various cell types. Indeed it is
known that at the level of the organ, oscillations are different. Central clock oscillation in the SCN is known
to oscillate in a different phase than peripheral tissues , whereby the SCN is phase advanced to the kidney,
[25]
which is phase advanced to aorta which is then phase advanced relative to liver . The arterial system
[26]
is composed of three layers; there is an adventitial layer (fibroblasts, pericytes, macrophages) , smooth
[27]
muscle cell layer (media), and an endothelial cell layer (endothelium). The current study examined clock
expression in mouse vascular tissue and the human saphenous vein. The approach we elected to use was
immunohistochemistry to study clock expression. One key advantage of immunohistological techniques is
the ability to differentiate cellular localization. We in particular were interested in endothelial vs. smooth
muscle, vs. the adventitia. To some extent, immunohistochemistry can also provide a rough estimate of
nuclear staining or extranuclear staining, but generally distinguishing between subcellular compartments
is limited by IHC. In terms of using a fluorescent secondary vs. a chromogenic secondary antibody, both
are useful, and generally there can be some background fluorescence in tissues with regard to lamina in
the vasculature, so we chose the chromogenic susbstrate reaction. In mouse, Bmal1 expression revealed
a very strong expression pattern at midnight in the adventitia, in the common carotid artery and aorta
and this adventitial staining was decreased at noon. Similarly, casein kinase and Npas2 were also highly
expressed in the adventitia. Conversely, media and endothelium staining for Bmal1 was stronger at noon,
suggesting that the vascular cell layers are uniquely controlled, which may reflect specific timing of the
individual cell-type but may also relate to coordination of paracrine signaling from cell-layer to cell layer.
In the femoral artery, adventitial staining was most striking for Npas2 and Cry1. Clock the heterodimeric
partner to Bmal1 was not as strongly expressed, but followed a similar temporal profile to Bmal1 in the
carotid and aorta in the media. In the heart, all clock components exhibited nominal staining at 12 AM,
but expression was robustly increased at 12 PM. In lung, the bronchiole epithelial cells were highly positive
for Rora. In the human saphenous vein, Bmal1 exhibited stronger expression at 12 PM vs. 12 AM, while
Per1 was in antiphase to Bmal1. Similarly, by western blotting, Bmal1 was antiphase to Cry1. Interestingly,
we have previously found that endothelial mechanisms such as eNOS and Akt follow or mirror circadian
clock expression in particular in regions of altered blood flow , while others have demonstrated that
[28]
eNOS follows the clock in aging . In the human veins, the clock was also expressed, and although blood
[29]
pressure is lower in the venous system than arterial, there is also evidence of a circadian rhythm in blood
pressure in the venous system . Another potential significance of the circadian clock in the venous
[30]
system is that it may also relate to disorders such as orthostatic hypotension. Orthostatic hypotension
has a prominent circadian component, which may relate to autonomic input dysfunction on both the
[31]
arterial and venous system, and interestingly has emerged as characteristic in Parkinson’s disease .
[32]
Thus the circadian system may be exerting different functions in the cells within the arterial system and
venous system, all which still is largely unknown. Our studies show that circadian clock components
display differences in expression and localization throughout the cardiovascular system, which may
confer nuances of circadian clock signaling in a cell-specific manner. The bloodstream is a key conduit
that relays biomechanical (hypertension) and biochemical information (hypercholesterolemia) from
environmental change or disturbance (jet lag, shift work, sleep dysfunction) to the vasculature, while there
may also be direct acting clock dampeners such as aging that act on endothelial cells directly to worsen
clock function [Figure 4]. These signals may impair function of the clock in ECs to impair other EC’s or to
impair SMCs, though it is still not clear if EC clocks communicate with SMC clocks and if there is even EC
to EC cell communication. Understanding oscillations of the clock in the cellular milieu of the vasculature
will be crucial in delineating how clocks can influence pathology of hypertension and atherosclerosis and
ultimately permit the development of improved therapeutic approaches that include timing and clocks into
maximizing efficacy and treatment.