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.
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.
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