TRPV channels belong to a superfamily of non-selective cation permeable channels, with a vast range of physiological functions. Our aim was to characterise the molecular and functional expression TRPV4 channels in retinal microvascular endothelial cells (RMECs) and to investigate their role in modulating retinal angiogenic signalling in vitro and in vivo. mRNA transcripts, protein expression and localisation of TRPV4 channels in cultured bovine RMECs were examined using RT-PCR, western blotting and immunocytochemistry, respectively. [Ca2+]i was monitored using Fura-2 microfluorimetry, in vitro angiogenesis using cell proliferation, migration, tubulogenesis and 3D sprouting assays and in vivo angiogenesis using the murine model of oxygen-induced retinopathy (OIR). Data were analysed using unpaired t-tests or ANOVA. “N” refers to number of biological replicates, “n” to the number of cells tested. mRNA and protein expression for TRPV4 was detected in RMECs (N=3). In immunocytochemistry studies TRPV4 channels were found to be mainly localised to the cytoplasm and cell nuclei, although some plasma membrane staining was also observed (N=3). The TRPV4 agonist GSK1016790A (100nM) elevated [Ca2+]i in RMECs (from a baseline R340/380 of 0.53 ± 0.02 to a peak of 2.41 ± 0.03; n=13,N=3), an effect that was blocked by pre-incubating the cells with the TRPV4 antagonist HC067047 (1μM) (p<0.001; n=8, N=3). HC067047, and a second TRPV4 inhibitor, RN1734, blocked in vitro sprouting angiogenesis in a concentration-dependent manner (IC50s for these drugs were 2.62μM ± 0.04707 and 3.2μM ± 0.06124, respectively), but had no effect on cell proliferation (Brdu ELISA) or cell migration (scratch-wound assay) (N=3, p>0.05 in both cases vs normal/DMSO controls). These drugs did, however, block tubulogenesis (HC067047 20μM, RN1734 15μM; N=2 per treatment). OIR was induced in C57BL6 mice as previously described (1). Mice were anesthetised at P15 with ketamine (60mg/kg) and xylazine (6mg/kg, both IP) and given 1µl intravitreal injections of TRPV4 inhibitors/control agents. At P17 animals were killed by schedule 1 methods, eyes were enucleated and retinas stained with isolectin B4 for quantification of avascular (AV), normal vascular (V) and neovascular (NV) areas. HC067047 (20μM; N=12, 200μM; N=5) and RN1734 (15μM; N=10, 150μM; N=5) reduced AV areas, increased V areas and decreased NV areas (p<0.05 for both drugs at all concentrations vs non-injected controls; N=9). Vehicle controls (0.01% DMSO) were without effect (p>0.05 for all parameters; N=6). These data suggest that TRPV4 plays a significant role in pathological angiogenesis in the retina by modulating endothelial tubulogenesis. TRPV4 inhibitors have potential therapeutic value for neovascular diseases of the eye.