P4501Identification and characterization of endothelial microRNAs that modulate leukocyte adhesion through novel mechanistic pathways

Abstract

Abstract Introduction Inflammation is essential for the protective response of the immune system. However, hyperactivated inflammation and dysregulated resolution strongly associates with the pathophysiology of atherosclerosis and ischemia-induced injury after myocardial infarction. Therefore, attenuation of inflammatory response has emerged as a promising approach to reduce cardiovascular disease burden. A limiting step of inflammation is the local recruitment of leukocytes to the lesion, a process regulated by intense cross-talk between immune and endothelial cells. A better understanding of the modulatory mechanisms of adhesion is paramount for the development of better therapies. Purpose Identify endothelial miRNAs that impact leukocyte adhesion and characterize the underlying pathways that regulate this process. Methods A functional high-throughput screening (HTS) of human miRNA libraries (mimics and inhibitors) measured miRNA impact on monocyte (THP-1) adhesion to an endothelial monolayer (HAEC). Individually miRNAs were transfected in HAEC and fluorescently-labeled monocyte attachment was recorded by a robotic automated microscopy platform. Computational analysis lead to identification of potential targets and relevant pathways associated to the action of candidate miRNAs. Further validation of promising targets was performed by qPCR and western blotting. Additional endothelial phenotypic properties such as cytoskeleton morphology or endothelial barrier function were analyzed in the presence of specific miRNAs. Results Functional HTS and secondary screening resulted in 38 microRNAs that reduced and 2 that increased monocyte adhesion. Bioinformatic target prediction and pathway analysis narrowed the set of miRNA candidates used for characterization studies. These miRNAs significantly modulated cell adhesion of both monocytic-leukemia THP-1 cells and freshly isolated human CD14+ monocytes, but effect on CD14+ was weaker compared to THP-1. Several miRNAs induced severe changes on endothelial cell morphology, likely due to cytoskeleton rearrangement. We identified and validated several miRNA targets belonging to the Ras GTPase family of actin remodeling modulators (RalA, RAP1A). Additionally, a few miRNAs targeted Ephrin signaling molecules (EFNs, EPHs) which mediate multiple cell functions including cell-cell contacts. We also explored miRNA effects on endothelial barrier function and measured monocyte adhesion under physiological and disturbed flow conditions. Conclusions We identified a set of miRNAs able to modulate monocyte cell adhesion to endothelial cells under inflammatory conditions. Potential mechanistic pathways of miRNA modulation of adhesion included Ephrin signaling pathway and Ras GTPase family. A better understanding of the role of specific microRNAs regulating the immune-endothelial cell interaction may lead to novel therapeutic strategies in atherosclerosis and myocardial infarction. Acknowledgement/Funding DZHK (German Centre for Cardiovascular Research), partner site Berlin, Germany; Berlin Institute of Health (BIH)