Abstract 501: A Pivotal Role of Smooth Muscle Bmal1 in Mineralocorticoid Receptor Agonist plus Salt Induced Mouse Aortic Aneurysm

Abstract

Rationale: Bmal1 is a basic helix loop helix (bHLH) transcription factor and is an indispensable core clock gene. Bmal1 is expressed in vascular smooth muscle and has been implicated in atherosclerosis and vascular remodeling. However, it is unknown whether vascular smooth muscle Bmal1 is involved in aortic aneurysm. We use a smooth muscle selective Bmal1 knockout (SM-Bmal1 -/- ) mouse and mineralocorticoid receptor agonists plus salt induced aortic aneurysm mouse model to address this specific question. Approach and Results: Aortic Aneurysm was induced in SM-Bmal1 -/- and littermate controls by subcutaneously implanting a 50mg DOCA (deoxycorticosterone acetate) pellet for 21 days or mini-pump with 200μg/Kg/day aldosterone for 28 days and drinking 0.9% saline solution. The abdominal aortic aneurysm incidence was dramatically decreased from control 30.7% (4-month-old) and 68.0% (8-month-old) to 4.0% (p<0.05) and 0.0% (p<0.001) in SM-Bmal1 -/- mice respectively. This was associated with a significant decrease in the aortic inner and external diameters. While SM-Bmal1 deletion did not alter the plasma sodium concentration, it significantly decreased the elastin breakage in aorta. In situ zymography assay demonstrated a significantly suppressed gelatinase activity in SM-Bmal1 -/- mouse aorta when the metalloproteinases (MMPs) were complexed with their tissue inhibitors (TIMPs). Gel zymography showed no decrease in MMP2/9 activities in the SM-Bmal1-KO mouse aorta, real-time PCR analysis showed TIMP4 mRNA and immunohistochemistry analysis showed TIMP4 protein were selectively up-regulated among the four TIMP isoforms. Mouse TIMP4 promoter contains 7 E-boxes, ChIP assay of the mouse aorta tissues showed 4 of them bound to Bmal1. Further TIMP4 promoter luciferase activity analysis demonstrated TIMP4 promoter activity was dramatically activated by the deletion of Bmal1. Conclusions: Deletion of Bmal1 from smooth muscle protects mice from mineralocorticoid receptor agonist plus salt induced aortic aneurysm at least in part via up-regulating TIMP4.