Immunohistochemistry of the circadian clock in mouse and human vascular tissues.

Ciprian B. Anea,Ana M. Merloiu,David J. R. Fulton,Vijay Patel,R. Dan Rudic

doi:10.20517/2574-1209.2018.46

Abstract

AimThe circadian clock is a molecular network that controls the body physiological rhythms. In blood vessels, the circadian clock components modulate vascular remodeling, blood pressure, and signaling. The goal in this study was to determine the pattern of expression of circadian clock proteins in the endothelium, smooth muscle, and adventitia of the vasculature of human and mouse tissues.MethodsImmunohistochemistry was performed in frozen sections of mouse aorta, common carotid artery, femoral artery, lung, and heart at 12 AM and 12 PM for Bmal1, Clock, Npas2, Per and other clock components. Studies of expression were also assessed in human saphenous vein both by immunoblotting and immunohistochemistry.ResultsIn this study, we identified the expression of Bmal1, Clock, Npas, Per1, Cry1, and accessory clock components by immunohistochemical staining in the endothelium, smooth muscle and adventitia of the mouse vasculature with differing temporal and cellular profiles depending on vasculature and tissue analyzed. The human saphenous vein also exhibited expression of clock genes that exhibited an oscillatory pattern in Bmal1 and Cry by immunoblotting.ConclusionThese 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.

Highlights

This circadian clock is a signaling mechanism that controls 24 h rhythmic oscillations
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]
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]

Summary

Introduction

This circadian clock is a signaling mechanism that controls 24 h rhythmic oscillations. In the circadian clock loop, the Bmal1-Clock heterodimer serves to cause transcription of Period and Cry genes which are translated to proteins, form heterodimers themselves, and cycle from the cytoplasm back into to the nucleus to inhibit Bmal and Clock. This molecular mechanism is expressed through the body, generating cyclic 24 h physiological rhythms. In cell specific knockout models of the intrinsic vasculature, smooth muscle disruption of Bmal results in alterations in rhythmic blood pressure[14], disruption of the circadian clock in the endothelium worsens the thrombogenic response and affects blood pressure[15], and vascular transplantation of Bmal1-KO mice into WT mice induces arteriosclerotic response[16]. We sought to examine the cellular expression of the circadian clock in the mouse and human vascular tissue

Objectives

Methods

Results

Conclusion