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Caitriona OLeary, Jennifer E McNaughten, Alan W Stitt, Graham J McGeown, Tim M Curtis; TRPV1 channels are functionally expressed in the retinal microvascular endothelium and contribute to retinal angiogenic signalling in vitro. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5373.
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© ARVO (1962-2015); The Authors (2016-present)
Transient receptor potential vanilloid (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 functional expression of calcium permeable Transient Receptor Potential Vanilloid (TRPV)-1 channels in retinal microvascular endothelial cells (RMECs) and to investigate their role in modulating retinal angiogenic signalling in vitro.
RMECs were isolated from bovine retinas and cultured with 10% porcine serum. mRNA and protein expression for TRPV1 were examined using RT-PCR and western blotting, respectively. The expression and sub-cellular localisation of TRPV1 channels in RMECs and blood vessels within bovine retinal wholemounts were assessed using immunohistochemistry. Intracellular Ca2+ concentration ([Ca2+]i) was monitored using Fura-2 microfluorimetry to test for the functional expression of TRPV1 channels in RMECs following treatment with TRPV1 specific activators and inhibitors. The effects of TRPV1 antagonists on retinal angiogenesis in vitro were assessed using a Matrigel sprouting assay.
Both TRPV1 mRNA and protein expression were identified in RMECs using RT-PCR and western blot analysis. In RMECs, TRPV1 was found to be mainly localised to the cytoplasm with some areas of staining on the cell membrane. TRPV1 was also apparent in the nucleus, in a staining pattern that closely resembled the nucleoplasmic reticulum. Staining patterns for TRPV1 in endothelial cells of bovine retinal vessels were similar, but more punctate than those observed for RMECs. The ultra-potent TRPV1 agonist, resiniferatoxin (10nM), caused a transient rise in [Ca2+]i in the majority of RMECs tested. These responses were abolished in the presence of two different TRPV1 antagonists, capsazepine (5μM) and AMG9810 (5μM). Inhibition of TRPV1 using capsazepine, AMG9810 or A7841681 resulted in a concentration-dependent reduction in the number of invading sprouts.
These data show that TRPV1 channels are functionally expressed in RMECs and could represent candidate targets for improved anti-angiogenic therapies for the eye.
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