During the course of this funding, we studied whether miRNA-loaded extracellular vesicles (EVs) released by neutrophils upon their activation with ANCA can target and be internalized by microvascular endothelial cells (ECs). In an in vitro model, EVs released by activated neutrophils were taken up very efficiently by ECs. This resulted in the transfer of three mature miRNAs to ECs, whose expression is normally very low or absent in quiescent ECs. The overexpression of two of these miRNAs in ECs resulted in profound endothelial damage, especially in inflammatory conditions, characterized by the induction of cell death, the inhibition of cell proliferation, and as a result, impairment of EC repair. The marked deleterious effect of one of these miRNAs on EC survival and repair could be explained in part by its capacity to inhibit the activation of several critical intracellular signaling pathways, as well as the expression of genes involved in cell migration and angiogenesis. In addition, we demonstrated that overexpression of these two miRNAs in ECs also resulted in induction of the expression of proinflammatory molecules.
Altogether, results from this work suggest a model whereby ANCA-activated neutrophils release EV that carry three miRNAs to microvascular EC, where their expression is normally very low or absent in resting state. The consequences of the induced expression of two of these miRNAs involve the induction of a pro-inflammatory response and direct microvascular damage, notably though the induction of EC death and the inhibition of endothelial repair, especially in inflammatory conditions. Thus, these results suggest that inhibition of these two miRNAs in EC may have a promising therapeutic potential for the protection of EC from neutrophil-mediated damage in AAV.
These results were presented through an oral presentation at the American Society of Nephrology Kidney Week in November 2017 (New Orleans, LA, USA), and through a poster presentation at the International Vascular Biology Meeting in June 2018 (Helsinki, Finland). A manuscript regrouping these data is currently under preparation.
During this fellowship, we also analysed miRNA expression profiles by TaqMan Low Density Arrays in neutrophils activated with ANCA in vitro. This led to the identification of 9 miRNAs that were systematically significantly deregulated upon ANCA activation. We then analysed the expression levels of these 9 miRNAs in neutrophils isolated from the blood of 10 patients with AAV either at the initial inflammatory phase at the time of diagnosis, or during the remission phase. This was done thanks to the set-up of a biocollection of samples from AAV patients, collected longitudinally throughout the evolution of their disease, in collaboration with the Internal Medicine Department at CHU de Nantes (NALVANCA biocollection). We thus identified two specific miRNAs that were increased at the time of diagnosis vs. remission. To achieve this work, we are currently enrolling additional patients and implementing the NALVANCA biocollection, in order to determine whether the expression of these miRNAs is increased in neutrophils at the time of a relapse and to increase the number of samples analysed to strengthen our results and perform statistical analyses. Eventually, this work should lead to another publication and a patent on identified miRNAs.
