br Acknowledgments br Introduction Hypertensive retinopathy

Acknowledgments
Introduction Hypertensive retinopathy is a disease of the retinal microvasculature associated with elevated blood pressure (Fraser-Bell et al., 2017). Narrowing of retinal arterioles occurs in the early stage of the disease, and with persistent hypertension further vascular pathology develops including vision-threatening breakdown of the blood-retinal barrier (BRB) (Fraser-Bell et al., 2017). Systemic hypertension is a risk factor for prevalent ocular diseases including age-related macular degeneration (Klein et al., 2003) and diabetic retinopathy (DR) (Klein et al., 1984a; b). Indeed, DR features damage to the retinal microvasculature (Fletcher et al., 2007) as well as the retinal pigment epithelium (RPE) and choroidal vasculature (Lutty, 2017). DR is a leading cause of vision loss and blindness in the working population throughout the world (Yau et al., 2012), yet preventative treatments are limited. As the global prevalence of diabetes increases to an estimated 642 million people by 2040 (Ogurtsova et al., 2017), there is an unmet medical need to identify new treatment targets to improve the outlook of patients with DR. The vasoactive pathway, the renin-angiotensin 4-Aminobutyric acid system, has a central role in regulating blood pressure and the development of DR. In particular, blood pressure lowering and blockade of angiotensin converting enzyme or the angiotensin type 1 receptor, improves DR in experimental models (Hamada et al., 1995; Miller et al., 2010; Phipps et al., 2007; Yamagishi et al., 2005) as well as retinopathy in subjects with type 1 and type 2 diabetes (Chaturvedi et al., 2008; Sjolie et al., 2011). However, these treatment approaches do not provide complete retinoprotection (Chaturvedi et al., 2008; Sjolie et al., 2011), suggesting that other vasoactive agents have a causal role. A potential candidate is endothelin (ET), a potent vasoconstrictor comprised of three isoforms designated ET-1, ET-2 and ET-3, whose actions are mediated by the ET type A receptor (ETRA) and ET type B receptor (ETRB) (Davenport et al., 2016; Yanagisawa et al., 1988). ET-1 and ET-2 bind to both receptors with similar affinity, while ET-3 binds only to the ETRB (Davenport et al., 2016). ET-1 is the most studied isoform due to its high expression in the vasculature, central role in the regulation of blood pressure (Davenport et al., 2016; Yanagisawa et al., 1988) and ability to influence a variety of cellular processes including cell proliferation and migration (Garrafa et al., 2012). ET-2 is less well studied due to its similar structure to ET-1, differing by only two amino acids in humans and three amino acids in rats and mice (Saida et al., 2000). Nonetheless, there is increasing interest in the actions of ET-2, which include the promotion of inflammation and vasculopathy (Alrashdi et al., 2017; Grimshaw et al., 2002). The development of ETRA and ETRB antagonists has assisted in understanding the contribution of ETs to physiological and pathological processes (Reichetzeder et al., 2014). Furthermore, inhibition of these receptors is of interest for the medical treatment of hypertension and diabetic complications, albeit systemic administration of ETRA inhibitors is controversial due to possible adverse cardiovascular events in patients (Mann et al., 2010; Reichetzeder et al., 2014). Components of the ET system are expressed in the retina (Chakravarthy et al., 1997; McDonald et al., 2010) including in patients with proliferative DR (Adamiec-Mroczek et al., 2010; Roldan-Pallares et al., 2005). However, whether individual antagonism of the ETRA and ETRB influence the development of DR as well as hypertensive DR has not been extensively explored. To determine if antagonism of the ET receptors improved DR independently of any anti-hypertensive effect, we used an intraocular route of administration. Spontaneously hypertensive rats (SHR) and their genetic controls, Wistar Kyoto rats (WKY), were made diabetic with streptozotocin, and we determined that ET transcripts and particularly ET-2 are increased in retina as well as RPE/choroid. Our findings demonstrate the ability of ETRA or ETRB antagonism to attenuate retinal vascular leakage and associated events in the presence of hypertension and diabetes.