A Cell-specific Endothelial MicroRNA Adenylation Pathway Regulates Th2 Inflammation in Asthma

Yale University, New Haven CT

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Abstract

PROJECT SUMMARY Asthma is one of the most common chronic disorders in the United States and an emergent disease worldwide. Airway vascular remodeling and increased expression of Vascular endothelial growth factor (VEGF) in lungs and serum of asthmatics correlate with the severity of their disease and the critical role of VEGF in type2 inflammation is also corroborated in animal studies. The mechanism of this contribution is not clear. We have previously shown that VEGF mediates miR-1 downregulation in the lung endothelium and this downregulation is critical for the expression of endothelial adhesion molecules and recruitment of inflammatory cells to the lung. Overexpression of miR-1 in endothelial cells inhibited VEGF mediated proliferation and differentiation. These findings suggested that lung endothelium is the main site of miR-1 regulation and that VEGF, directly and specifically regulates this intrinsic endothelial pathway. We have generated a vascular- specific miR-1 lentiviral vector, and an inducible vascular-specific miR-1 transgenic mouse model to test this endothelial specificity. In preliminary studies delivery of the vascular-specific lentiviral vector inhibited ovalbumin-induced eosinophilia and cytokine release, suggesting that low endothelial miR-1 level is necessary for the propagation of Th2 inflammation. In our preliminary studies on the mechanism of VEGF-mediated miR- 1 downregulation we discovered that VEGF decreases mature miR-1 without lowering its precursors or its binding to Argonaute 2 protein. VEGF stimulation also induced the emergence of unique 3'-adenylated and trimmed miR-1 isoforms (isomiRs) in endothelial cells, suggesting that miR-1 adenylation triggers its degradation. Furthermore, knockdown of a poly(A) polymerase enzymes responsible for miRNA adenylation, increased miR-1 levels and inhibited VEGF-mediated endothelial cell proliferation. Based on our preliminary results we hypothesize that VEGF mediates its effect on airway inflammation through specific adenylation and degradation of miR-1 in the lung endothelial cells. In aim1 we will characterize the miR-1 isoforms induced by VEGF and Th2 inflammation in the lung and airway endothelium and characterize the kinetics of miR-1 decay and adenylation, the role of miR-1 sequence, and the site of these modification within the endothelial cells. In aim2 we will test the effect of vascular-specific miR-1 overexpression by lentiviral delivery or transgenic overexpression in VEGF transgenic and Th2 inflammation models and also analyze the effect of poly A polymerase knockdown in these models. In aim3 we will examine the correlation ofmiR-1 and its adenylation pathway with the severity of type2 inflammation in asthma.