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Introduction Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid principally derived from oily fish, has been shown to reduce the incidence of cardiovascular events and related mortality. These benefits are attributed in part to its ability to lower circulating triglyceride concentration and to exert potent anti-inflammatory and cytoprotective effects. The vasculoprotective actions of DHA may arise through activation of lipid-sensing nuclear receptors, G-protein-coupled receptors (GPCRs), incorporation into cellular phospholipid membranes, or the generation of bioactive oxygenated metabolites, such as the specialised pro-resolving mediator (SPM) resolvin D1 (RvD1) and its precursor 17-HDHA. In vitro studies have shown that 17-HDHA and RvD1 can limit leukocyte activation, suppress inflammatory signalling, and promote resolution of inflammation, largely through GPCRs. However, their precise actions on the vasculature remain poorly defined and whether, and how, they directly target endothelial cells remains elusive. This study evaluated the direct effects of 17-HDHA and RvD1 on human endothelial cell functions in vitro.Methods/Results Human umbilical cord endothelial cells (HUVEC) were treated with RvD1 and 17-HDHA and their angiogenic potential was assessed using 2D tube formation and 3D co-culture angiogenesis assays. Both RvD1 (50nM) and 17-HDHA (100nM) significantly enhanced tube and vessel formation, respectively, as well as the number of nodes and total tubule length ( figure 1). Neither 17-HDHA nor RvD1 affected HUVEC migration or proliferation as assessed using a combination of functional assays (MTS, BrdU, scratch wound assay). In adult human cardiac microvascular endothelial cells (HCMEC) treated with TNFα (2ng/ml), 17-HDHA restored peripheral cytoplasmic integrity by reinstating ZO-1 protein expression on the cell surface tight junctions. Additionally, in an orbital shaker flow model using human aortic ECs (plated on biotinylated gelatin), 72 hours of disturbed flow caused an increase in monolayer permeability (measured using fluorescently labelled streptavidin), and this was significantly reduced by treatment with RvD1. Gene expression analysis using qRT-PCR was performed to measure expression of GPCRs previously implicated in mediating the effects of DHA-derived SPMs (FPR2, FPR3, GPR18 and GPR32). These studies showed that FPR3 was highly expressed in all EC types used for the functional studies whereas expression of FRP2, GPR18 and GPR32 was very low or absent. RvD1 and 17-HDHA-driven 2D tubulogenesis was blocked by WRW4 (10μM), a non-selective FPR2 and FPR3 antagonist, showing that FPR2/3 receptors are indispensable. RvD1 and 17-HDHA enhanced phosphorylation of ERK1/2 in HUVEC and HCMEC, and PD184352 (10μM), a MEK1/2 inhibitor, significantly reduced RvD1- and 17-HDHA-driven 2D tube formation. To test whether these DHA-derived lipid mediators exert pro-repair effects in a model with clinical relevance, an established zebrafish model of cryoinjury-induced myocardial infarction was employed. A single IP dose of RvD1 (2μg/kg), 17-HDHA (4μg/kg) or ethanol (0.2%) was administered to Tg(fli1a:DsRedex) zebrafish prior to cryoinjury (Chablais et al, 2011). Immunofluorescence analysis of the hearts at 7 days post-cryoinjury showed that 17-HDHA supplementation increased expression of fli1a endothelial marker, suggesting enhanced endothelial cell proliferation within the injury area to support vascular repair.Conclusion These results demonstrate that 17-HDHA and RvD1 exert pro-angiogenic and cytoprotective actions in primary human endothelial cells and may contribute to vascular pro-reparative mechanisms upon injury or inflammation-related stress.Abstract 307 Figure 117-HDHA and RvD1 promote vessel formation in a 3D co-culture angiogenesis assay. HUVEC were plated on top of a confluent monolayer of primary human dermal fibroblasts and cultured in the presence of 17-HDHA (100nM), RvD1 (50nM), VEGF (20ng/ml) and EtOH (0.1%) as vehicle control for a total of 7 days. The cultures were then fixed, stained with an anti-CD31 antibody (green) and the formation of 3D vessel-like structures was captured using an inverted fluorescence microscope. Image analysis was performed using the Image-Pro Angiogenesis protocol. Data are from 3 individual experiments in 3 cell isolates with each point representing an imaged area (mean ± SEM), ***p<0.001, ****p<0.0001.