Endothelial cell (EC) activation and dysfunction have been linked to a variety of vascular inflammatory disease states including atherosclerosis-the major cause of morbidity and mortality in Western Societies. Accumulating studies highlight a critical role for enhanced NF-kB pathway activation in atherosclerotic tissues including the vascular endothelium. Proinflammatory cytokines and proatherogenic risk factors such as hyperlipidemia lead to NF-kB activation, an effect that confers pro-adhesive, pro-thrombotic properties to ECs. Therefore, suppressing the inflammatory response in the vascular endothelium may provide a novel therapeutic approach to limit atherothrombosis. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs capable of repressing gene expression by base pairing to the 3'untranslated regions (3'-UTRs) of mRNA targets and are involved in a variety of pathophysiological processes including the regulation of immune and inflammatory responses. However, the role of microRNAs in atherosclerotic-associated endothelial activation remains poorly defined. We undertook a microarray profiling approach in endothelial cells (ECs) and identified that miR-181b expression is rapidly reduced in response to TNF-a and in the vascular endothelium from atherosclerotic-prone mice - observations that are recapitulated in human inflammatory paradigms in vivo. Based on our preliminary studies, we find that miR-181b overexpression inhibited TNF-a-induced NF-kB-responsive targets including adhesion molecules (e.g. VCAM-1, E-selectin), chemokines (e.g. CX3CL1, CXCL1), and proinflammatory mediators (e.g. COX-2, PAI-1) that are critical to lesion formation, composition, or pre-disposition to thrombosis. Gain and loss-of function studies reveal that miR-181b potently inhibited leukocyte adhesion to endothelial monolayers, whereas inhibition of miR-181b had the opposite effect. Mechanistically, we find that MiR-181b suppressed the activation of the NF-?B pathway by binding uniquely to the 3'UTR of importin-a3, a protein involved in the nuclear translocation of NF-?B, and reduced its expression. Finally, systemic intravenous administration of miR-181b mimics reduced EC activation and leukocyte accumulation in vivo. These observations provide the foundation for the central hypothesis that miR-181b may serve as a critical regulator of EC activation and vascular homeostasis. To better understand the precise role of miR-181b in NF-kB signaling and EC activation, three aims are proposed.
In Aim1, we will delineate the upstream mechanisms governing miR-181b expression in ECs.
In Aim 2, we will determine the molecular basis for miR-181b's ability to regulate NF-kB signaling and EC activation.
In Aim 3, we will explore the effect of altered miR-181b expression on acute and chronic vascular inflammation. The results of these studies will provide insights regarding miR-181b function in EC biology, vascular inflammation, and atherothrombosis and may provide new targets for anti-inflammatory therapy.

Public Health Relevance

The activation of the vascular endothelium is a characteristic feature seen in atherosclerosis and other chronic inflammatory diseases, an effect that may promote disease progression leading to heart attack, stroke, or peripheral vascular disease. We have identified a novel microRNA, termed miR- 181b, that is rapidly reduced in response to pro-inflammatory cytokines in endothelial cells and our studies indicate that miR-181b may act to dampen vascular inflammation within the vessel wall. The proposed studies will provide a detailed understanding underlying the function of this factor with the goal of developing novel therapies for the treatment of acute and chronic inflammatory disease.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01HL115141-03
Application #
8678992
Study Section
Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
Program Officer
Kirby, Ruth
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Brigham and Women's Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02115
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