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

INTRODUCTION
This circadian clock is a signaling mechanism that controls 24 h rhythmic oscillations. Heterodimeric
interactions of clock transcription factors including Clock, Npas2 with Bmal1 bind E-box response elements
of respective genes and drive transcription of downstream negative clock regulators or putative output
genes that regulate physiologic function. In the circadian clock loop, the Bmal1-Clock heterodimer serves
to cause transcription of Period and Cry genes which are then translated to proteins, form heterodimers
themselves, and cycle from the cytoplasm back into to the nucleus to inhibit Bmal1 and Clock. This
molecular mechanism is expressed through the body, generating cyclic 24 h physiological rhythms. In
blood vessels, the circadian clock is also oscillating . Since blood vessels are comprised of three distinct
[1-3]
cellular layers, the endothelial cell layer, smooth muscle cell layer, and the adventitial layer, circadian clocks
may exert cell specific and cell-coordinated functional actions intrinsic within the vasculature. Evidence
of vascular cell-specific functions of the circadian clock has first been demonstrated in global knockout
models. Global disruption of Bmal1 or Period genes impairs endothelial function and detrimentally
influences the adaptation of the vasculature both acutely and chronically . While the circadian clock is a
[4-9]
resilient or robust signaling pathway whereby its disruption is not lethal due to redundancy, intercellular,
and intracellular coupling [10,11] , overexpression of clock components has been shown to offer protection
against deleterious vascular phenotypes [12,13] . In cell specific knockout models of the intrinsic vasculature,
smooth muscle disruption of Bmal1 results in alterations in rhythmic blood pressure , disruption of the
[14]
circadian clock in the endothelium worsens the thrombogenic response and also affects blood pressure ,
[15]
and vascular transplantation of Bmal1-KO mice into WT mice induces arteriosclerotic response . In the
[16]
current study, we sought to examine the cellular expression of the circadian clock in the mouse and human
vascular tissue.

METHODS
Animals
All animal studies were performed according to protocols approved by the Medical College of Georgia
Institutional Committee for Use and Care of Laboratory Animals at Augusta University. Normal wild type
C57BL6 mice were used in all experiments.

Human blood vessels
Segments of intact human saphenous vein were obtained as discarded tissue from other surgical procedures.
The procurement of these tissues conforms to the principles outlined in the Declaration of Helsinki and was
approved by the human assurances committee of the Augusta University.

Materials
Tissue sections were probed using the following Antibodies: polyclonal for Bmal1 (Affinty Bioreagents),
monoclonal for Clock (Santa Cruz), monoclonal for Npas2 (Abnova), polyclonal for Per1 (Affinty Bioreagents),
polyclonal for Cry1 (Novus Biologicals), polyclonal for Rev-erbα (Cell Signaling Technology), polyclonal
for Rora (Cell Signaling Technology), monoclonal for Ck1-e (Bectin Dickinson), and polyclonal antibody
for Epas (Novus Biologicals), to determine protein expression and localization within the blood vessel.

Western blotting
Excess tissue from human saphenous veins from patients undergoing coronary arterial bypass surgeries
(CABG) were transferred to a dish and kept in EBM2 media (Lonza) in the incubator at 37 ºC for further
processing at serial time points. Vessels were pooled to permit detection of specific proteins, pulverized on
liquid nitrogen, and then immersed into protein lysis buffer.