Apart from the ET system s role in regulating
Apart from the ET system's role in regulating blood vessel tone, it is also involved in a variety of cellular processes that are relevant to DR. For example, the ET receptors influence the survival of retinal ganglion Z-DQMD-FMK (McGrady et al., 2017; Tonari et al., 2012), development of the retinal vasculature (Rattner et al., 2013) and are involved in the cytotoxic swelling of retinal Müller cells (Vogler et al., 2016). Inflammation has emerged as an important contributor to the development of retinal vascular disease (Tang and Kern, 2011). Our data suggest that ET may promote the inflammatory state of the retina with both BQ123 and BQ788 attenuating the increase in retinal ICAM-1, TNFα and MCP-1 in hypertension and diabetes. These data are consistent with a previous study demonstrating that antagonism of the ETRA reduced ICAM-1 expression in retina of normotensive and diabetic Sprague Dawley rats (Masuzawa et al., 2006). The effect of ETRB inhibition in the retina has been less studied, albeit BQ788 reduced TNFα levels in a model of optic nerve crush (Tonari et al., 2012) and we have previously reported that BQ788 attenuated ET-2-mediated infiltration of macrophages into the retina (Alrashdi et al., 2017). Overall, these findings contribute to the emerging pro-inflammatory actions of ET in tissues including the retina. Our findings extend the functions of the ETRA and ETRB to an involvement in the integrity of the BRB in hypertension and DR, with BQ123 and BQ788 attenuating retinal vascular leakage. Breakdown of the BRB is influenced by the health of macroglial Müller cells associated with the vasculature (Bringmann et al., 2006), and relevant to this study is the finding that this cell population expresses both the ETRA and ETRB (Vogler et al., 2016). A sign of Müller cell stress in diabetes is their heightened expression of GFAP as well as their increased production of VEGF (Bringmann et al., 2006; Murata et al., 1996). Indeed, this excessive production of VEGF compromises the BRB, and therefore VEGF is a major target for the treatment of proliferative DR and diabetic macular oedema (Simo and Hernandez, 2015). Here, we demonstrated that hypertension induced similar effects as diabetes on GFAP immunolabeling in Müller cells and VEGF levels in the retina and also increased VEGF expression in the RPE/choroid. The RPE is a major source of VEGF (Saint-Geniez et al., 2006), and the increased production of VEGF at this site increases the permeability of the RPE (Ablonczy and Crosson, 2007; Desjardins et al., 2016). Of significance was the additive effect of diabetes on these parameters, findings that highlight the consequences of systemic hypertension on the diabetic retina. The utility of ET receptor blockade as a treatment for hypertensive retinopathy and DR has not been extensively studied, although there are reports that the systemic administration of an ETRA antagonist reduced retinal VEGF levels and a combined ETRA/ETRB antagonist attenuated retinal vascular pathology in normotensive diabetic animals (Chou et al., 2014; Masuzawa et al., 2006). The mechanisms by which ET receptor blockade reduces VEGF levels in the retina and RPE/choroid are not fully understood. In other tissues, interactions between ETs and their receptors induces VEGF via activating integrin-linked kinase, Akt and hypoxia-inducible factor-1α signaling cascades (Spinella et al., 2010; Wu et al., 2014), events that can be attenuated by ET receptor blockade. Of interest, is whether these mechanisms are operating in DR, and if ET receptor blockade can attenuate the increase in VEGF levels and vascular permeability to a similar extent as anti-VEGF agents. A novel aspect of our study was that the local rather than the systemic administration of individual antagonists of the ET receptors effectively ameliorated the aforementioned parameters in the diabetic retina and RPE/choroid including in the presence of elevated blood pressure. These findings suggest that the local ocular ET system may influence vision-threatening events, albeit further study is required to make firm conclusions. Furthermore, the lowering of elevated blood pressure may not in all instances be a pre-requisite for improvements in DR since benefits were also seen in normotensive animals. However, this retinoprotection afforded by ETRA and ETRB antagonism requires further evaluation to determine if these treatment approaches ameliorate other pathological features of DR including damage to neurons (Fletcher et al., 2007).