br Methods br Results br
Discussion The findings of this study indicate that an ET system including ET-1 and ET-2 as well as ET receptors are present in the retina and RPE/choroid, and are up-regulated by both hypertension and diabetes. Specifically, individual antagonism of the ETRA or ETRB delivered locally into the eye, reduced vision-threatening vascular leakage and associated GFAP immunolabeling as well as the increased Sodium ascorbate of VEGF and inflammatory factors within the retina. Further, the RPE/choroid which can become damaged in DR (Lutty, 2017), also responded to blockade of the ETRA and ETRB with a reduction in VEGF. A novel aspect of our study was that intravitreal administration rather than systemic administration of the ET receptor antagonists conferred ocular protection, and despite the elevated blood pressure of the SHR. This finding may be important when considering the complex systemic actions of the ETRA and ETRB (Davenport et al., 2016). A major function of the ETRA is vasoconstriction (Davenport et al., 2016) while the ETRB is a potent vasodilator, acts as a clearance receptor for ET and promotes sodium retention in the kidney medulla (Davenport et al., 2016). Therefore, to avoid unwanted side effects, ETRA antagonists rather than ETRB antagonists have been most widely studied. However, depending on the selectivity of the ETRA antagonist for the ETRA and ETRB, the doses used and the presence of kidney disease, systemic administration of ETRA antagonists can induce sodium retention and oedema (Baltatu et al., 2012; Smolander et al., 2009). Indeed, in a clinical trial of patients with type 2 diabetes and overt nephropathy, although oral administration of the ETRA antagonist avosentan reduced proteinuria when added to standard treatment, avosentan also caused significant sodium overload and congestive heart failure in some participants resulting in termination of the study (Mann et al., 2010). Our data when placed in the context of patients with hypertension or diabetes which often experience advanced renal disease, as well as diabetic patients in which kidney and retinal disease often cluster (Parving et al., 1988), indicate that ocular administration of BQ123 and BQ788 might be of benefit for DR. A striking finding was that hypertension or diabetes increased ETRA, ETRB, ET-1 and ET-2 expression in retina and RPE/choroid. These levels were not further increased by the combination of hypertension and diabetes indicating that either condition may have induced maximal stimulation of the ET system at these sites. The increased ET-1 expression in diabetic retina is consistent with earlier reports (Chakravarthy et al., 1997; De Juan et al., 2000; Deng et al., 1999), albeit reduced ET-1 mRNA levels have been detected in retina of non-diabetic SHR (McDonald et al., 2010). The reasons for the differences between that study and ours is unclear, but may be due to age-associated damage to the retina, with the previous study evaluating 21-month-old SHR (McDonald et al., 2010), while our studies were performed in 3.5-month-old SHR. The role of ET-2 in ocular disease is not fully defined, although growing evidence indicates its expression is induced in various situations of retinal stress including glaucoma (Howell et al., 2011) and retinal and photoreceptor degeneration (Bramall et al., 2013; Rattner and Nathans, 2005; Samardzija et al., 2012) where it may have a neuroprotective role. Our findings indicate that ET-2 was markedly increased in both retina and RPE/choroid by hypertension and diabetes, and to a greater extent than ET-1. Our recent report indicates that rather than having a protective role, ET-2 can injure the retina, with intravitreal administration inducing retinal pathology reminiscent of DR including vascular leakage and inflammation (Alrashdi et al., 2017). The findings of the present study are consistent with this idea, with blockade of the ETRA (BQ123) effectively reducing these events as well as the elevated levels of ET-1 and ET-2 in retina and RPE/choroid. However, although blockade of the ETRB (BQ788) reduced ET-2 in both locations and ET-1 in RPE/choroid, it did not significantly reduce ET-1 in WKY or SHR retina. These data suggest that ETRA blockade is sufficient to suppress an overactive ET system in the hypertensive and diabetic retina. The mechanisms by which this occurs are not fully understood, but we speculate that the beneficial effect of ET receptor blockade on cells within the retina and RPE/choroid may have reduced their increased production of ET-1 and ET-2. A limitation of our study was that we were unable to further interrogate these findings by measuring the protein levels of ET-1 and ET-2 due to the absence of specific antibodies that distinguish between these similar ET isoforms.