Angiotensin receptors in the eyes of arterial hypertensive rats

Introduction

The renin-angiotensin system (RAS) is well known to be involved in regulating systemic blood pressure via the control of sodium balance, body fluid volumes and vascular tone (Hall 2003; Jackson 2006). In addition to the circulating RAS, there is a specific tissue-localized RAS (Kramkowski et al. 2006; Paul et al. 2006) that regulates long-term changes in various organs (Deschepper et al. 1986; Derkx et al. 1987) including the eye (Sramek et al. 1992; Danser et al. 1994; van Haeringen 1996; Wagner et al. 1996; Savaskan et al. 2004).

Drugs involved in RAS have been shown to be able to reduce intraocular pressure (IOP) in animal (Watkins et al. 1987; Giardina et al. 1990; Shah et al. 2000; Inoue et al. 2001; Wang et al. 2005) and human studies (Costagliola et al. 1995, 2000). Our study group has very recently found that intravitreally administered Angiotensin (1–7) [Ang (1–7)], a breakdown product of Angiotensin II (Ang II), reduces IOP even in the normotensive rabbit eye (Vaajanen et al. 2008a). In addition to these IOP-lowering effects, drugs involved in RAS have been reported to protect against diabetic retinopathy (EUCLIC Study Group 1997; Sjølie & Chaturvedi 2002).

The effects of angiotensins are exerted through specific heptahelical G-protein-coupled receptors (de Gasparo et al. 2000; Jackson 2006). Ang II receptors in the cardiovascular system are divided into three main subtypes: Ang II type 1 (AT₁), type 2 (AT₂) and type 4 (AT₄) receptors. In addition to these three classical receptor types, there is the recently discovered Mas receptor. It is also a G-protein-coupled receptor encoded by the Mas protooncogene. It mediates a number of the positive cardiovascular effects of Ang (1–7) – e.g. vasodilatation, antiproliferation, antifibrosis – and it has a counter-regulatory role in fluid volume homeostasis for Ang II and AT₁ activation (Santos et al. 2000, 2003).

The main aim of the present study was to establish the presence of the novel Mas receptor for vasorelaxing and antiproliferative peptide Ang (1–7) by using reverse transcriptase polymerase chain reaction (RT-PCR). The other aim was to quantify the expression profile of the AT₁ and AT₂ receptors in the ocular tissues of two types of arterial hypertensive rats and their respective controls with or without antihypertensive treatment by using an autoradiography method.

Materials and Methods

Animals

The eyes used here were obtained from animals involved in our previous study (Vaajanen et al. 2008b), in which the relationship between the development of hypertension and IOP was investigated using two arterial hypertensive rat strains and their normotensive control animals. The hypertensive animals were: male spontaneously hypertensive rats (SHRs) purchased from Charles-River (Charles River Wiga GmbH, Sulzfeld, Germany) aged 15 weeks and weighing 308 ± 5 g [mean ± standard error of the mean (SEM), n = 8]; and double transgenic rats harbouring human renin and human angiotensinogen genes (dTGR; Biotechnology and Animal Breeding Division, Füllingsdorf, Switzerland), aged 7 weeks and weighing 236 ± 6 g (mean ± SEM, n = 8). Their respective age-matched arterial normotensive controls were Wistar Kyoto (WKY; Charles River Wiga GmbH) weighing 350 ± 13 g (mean ± SEM, n = 8) and Sprague Dawley rats (SD; Taconic Europe A/S, Ry, Denmark) weighing 286 ± 5 g (mean ± SEM, n = 8). Half of each group was treated with oral AT₁ receptor antagonist: olmesartan medoximil 10 mg/kg/day (Olmetec^®; Leiras, Turku, Finland) for SHR and their WKY controls and valsartan 30 mg/kg/day (Diovan^®; Novartis, Basel, Switzerland) for dTGR and their SD controls. Two different ‘sartans’ were used because dTGR and their controls were obtained from another, concomitant (cardiovascular) study carried out by our research group in addition to the original study of SHR and WKY rats, and treated according to a study protocol of their own. Tablets were ground and mixed in the food. At the end of the treatment (10 weeks for SHR and WKY and 3 weeks for dTGR and SD) the rats were killed with CO₂/O₂ (AGA, Riihimäki, Finland) and decapitated. The short lifespan and early death of dTGR because of severe hypertension and its complications were the reasons for the difference in ages and duration of antihypertensive treatment.

In our previous in vivo study with these same rats, a slight positive relationship (p ≤ 0.05) between developing blood pressure and IOP in SHR was seen during 8 weeks’ follow-up, while the high baseline IOP in arterial hypertensive dTGR fell drastically in their short follow-up period. At the end of that in vivo study the eyes were enucleated immediately after death and snap-frozen in isopentane at −40° and stored at −70° for RT-PCR analysis and in vitro autoradiography. Blood pressure and IOP in each rat group prior to taking eye samples are shown in Fig. 1.

Real-time quantitative RT-PCR

Enucleated whole frozen eyes were embedded in OCT^™ compound (Tissue-Tec^®, Sakura, Japan) cornea upwards and 150 μm vertical sections of the whole eye were cut using a cryostat (−19°). The presence of mRNA of AT_1a, AT₂ and Mas receptors was determined at the level of the ciliary body and the level of the anterior retina without ciliary tissue (Fig. 2).

RNA extraction and cDNA synthesis from tissue sections were performed as described previously (Lakkisto et al. 2002). The expression of AT_1a, AT₂ and Mas receptor mRNAs and housekeeping control 18S were studied by real-time quantitative RT-PCR using the ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, California, USA). The following primer sequences were used: AT_1a forward 5′-GGCAGCCTCTGACTAAATGGC-3′ and reverse 5′-ACGGCTTTGCTTGGTTACTCC-3′; AT₂ forward 5′-TGTCTGTCCTCATTGCCAACA-3′, reverse 5′-TTCATTAAGGCAATCCCAGCA-3′ and probe 5′FAM-TCAGAACCATTGAATACTT-MGB; and Mas forward 5′-TCATGTGTATTGACAGCGGAGAA-3′ and reverse 5′-CACTAACATGAGCGGAGTGAAGA-3′. PCR reactions for AT_1a and Mas receptors were performed in duplicate in a 25-μl final volume containing 1X SYBR Green Master mix (Applied Biosystems) and 300 nm of primers. PCR reactions for AT₂ receptor and 18S were performed in duplicate in a 25-μl final volume containing 1X TaqMan Master mix (Applied Biosystems) and 300 nm of AT₂ receptor primers and 150 nm of AT₂ probe, or 1X 18S TaqMan Gene Expression Assay primer and probe mix (Hs999999_s1, Applied Biosystems). PCR cycling conditions were 10 min at +95° and 40 cycles of 20 seconds at +95° and 1 min at +60°. Data were analysed using the absolute standard curve method, as described elsewhere (Lakkisto et al. 2002). 18S was used for normalization of results.

Results

RT-PCR

Overall, the mRNA expression of AT_1a, AT₂ and Mas receptors was markedly higher in the retina than in the ciliary body in every rat group (Fig. 3). There was no clear difference in AT_1a receptor expression between different rat strains, but hypertensive dTGR had higher receptor expression compared to SHR. The expression of the novel Mas receptor was lower than that of AT_1a, but higher than that of AT₂ receptors in all groups except WKY rats.

Because treatment with ARB did not systematically influence the expression of mRNA for Mas receptors or that of AT_1a or AT₂ receptors, the results from non-treated and treated rat groups were combined for RT-PCR analysis. Only treated SHR animals evinced slightly but not significantly lowered expression of AT_1a receptors in both tissues evaluated.

Autoradiography

The densities of AT₁ and AT₁ receptors in the retina were overall higher compared to the respective ciliaris in every rat group, in accordance with the results of the RT-PCR analysis (Fig. 4A). There was no clear difference in AT₁ receptor densities between different rat strains, but ARB-medicated animals had lower AT₁ density, especially in hypertensive dTGR. The density of AT₂ receptors was overall smaller than that of AT₁ receptors in every rat group (Fig. 4B). An example from the autoradiography analysis is presented in Fig. 5.

A difference was seen between ARB-treated and non-treated animals in this analysis. The apparent density of AT₁ receptors in the retina in treated animals was approximately 30% lower in SHR and SD rats and about 60% lower in dTGR and WKY compared with non-treated animals. This tendency was also seen in the ciliary body. Densities of AT₁ and AT₂ receptors and the respective blood pressure values in individual animals are presented in Fig. 6. The results suggest that the density of AT₁ receptors in the eye is independent of the blood pressure level of the animals, but more dependent on oral ARB treatment. Medicated dTGR and WKY had significantly lower retinal AT₁ density compared to the non-treated animals. Medication with ARB did not systematically influence the density of AT₂ receptors but medicated dTGR had more AT₂ receptors, especially in the retina (p < 0.05).

Discussion

The present study focuses on angiotensin receptors in the eyes of arterial hypertensive animals. Abnormal blood pressure is a known risk factor for some ocular diseases such as glaucoma (Tielsch et al. 1995; Bonomi et al. 2000) and retinopathy (Luo & Brown 2004; Walsh et al. 2006), although the exact pathophysiological mechanism underlying these disorders is unclear. Hypertension has been reported to be related to high IOP (Dielemans et al. 1995), although observations to the contrary have also been reported (Leske et al. 1995). According to the vascular theory, blood pressure and especially low perfusion pressure are linked to the progression of glaucoma damage (Flammer et al. 2002). High blood pressure can cause a number of direct vascular changes in the retina, leading to poor vision (Walsh et al. 2006).

The RT-PCR results of this study indicate for the first time the presence of the novel Mas receptor in eye tissue. The Mas receptor and AT_1a and AT₂ receptors were found in all rat strains, being more pronounced at the level of the retina than that of the ciliary body. mRNA of these receptors was determined from slices cut from a whole eyeball at the level of the ciliary body and at the level of the retina without ciliary tissue. It was thus not possible to establish more specifically the type of cells in which each receptor was localized. Interestingly, the retinal mRNA expression of all angiotensin receptors was higher in dTGR than in SHR, while the arterial normotensive control animals also yielded high values of mRNA of AT₁ receptors, indicating a strain-dependent variation of as yet unclear significance. No clear effect of oral antihypertensive treatment on mRNA expression of receptors was observed in RT-PCR analysis. According to other investigators, e.g. Zhou et al. (2005, 2006), treatment with AT₁ receptor antagonists blocked (cerebrovascular) AT₁ receptors in SHR with no significant effect on receptor protein and mRNA expression. These data support our decision to combine the results of untreated and treated animals in RT-PCR analysis.

The autoradiography results suggest that the receptor densities in the eye were independent of the blood pressure level of the animals, but more dependent on oral ARB treatment. The AT₁ receptor blockade in treated animals was most obvious in the retina of dTGR, but the same tendency was also seen in other medicated rats. ARB treatment did not influence AT₂ receptor density systematically but medicated dTGR had higher receptor levels, especially in the retina. Observations consistent with this (Zhou et al. 2006) but also contrary (Nishida et al. 2008) have been reported. There was a big difference between results of mRNA expression and ‘true’ receptor density. We have no clear explanation for this, but the RT-PCR method measures mRNA for receptors in the tissues so the ‘tools for receptors’ were found in all rat strains in retina and ciliaris (although in the case of receptor density the differences were less obvious). The density of the novel Mas receptor could not be examined by in vitro autoradiography because of a lacking methodology.

Ang II receptors are classically divided into three main subtypes. Most known biological effects of vasoactive Ang II are mediated by the AT₁ receptors in cardiovascular and other target cells, which are specifically blocked by AT₁ receptor antagonists, widely used as antihypertensive drugs –‘sartans’ (de Gasparo et al. 2000; Burnier 2001). In rodents, AT₁ receptors are further divided into AT_1a and AT_1b receptors, having similarities in their ligand binding and activation properties but differences in their tissue distribution (Kakar et al. 1992; de Gasparo et al. 2000). There is previous evidence regarding the expression of AT_1a in rat ocular tissue (Murata et al. 1997; Wheeler-Schilling et al. 1999; Mizoue et al. 2006). AT₂ receptors are characterized less well than AT₁ receptors, but they are considered as cardiovascular-protective receptors that antagonize the effects of Ang II mediated via AT₁ receptors. They have been identified in eyes in only a few studies (Senanayake et al. 2007). AT₄ receptors are not determined in detail, but they are known to be involved in cardiovascular pathology (Ruiz-Ortega et al. 2007). In addition to these three classical receptor types there is the recently discovered Mas receptor, which has now been determined in ocular tissue for the first time.

The Mas receptor was found a couple of years ago, first in the mouse kidney and subsequently in other organs (e.g. heart, brain and vasculature) (Santos et al. 2003; Iwata et al. 2005). In vivo Mas receptor acts as an antagonist to the AT₁ receptor, and in addition Mas can hetero-oligomerize with the AT₁ receptor and by doing so inhibit the actions of Ang II (Kostenis et al. 2005). There are no previous reports concerning Mas receptor expression in ocular tissues, although our recent functional study using Mas receptor agonist, Ang (1–7), and antagonist suggested its existence (Vaajanen et al. 2008a, 2008c). Ang (1–7), a heptapeptide formed from Ang II (Welches et al. 1993; Brosnihan et al. 1996; Muthalif et al. 1998; Santos et al.2000; Kucharewicz et al. 2002), is an endogenous ligand for this receptor type (Santos et al. 2003). It has been found very recently in the human retina (Senanayake et al. 2007).

To summarize, we have previously found a functional Mas receptor when intravitreally administered Ang (1–7) lowered IOP in rabbits. This effect was abolished by A-779 (=Mas receptor antagonist) (Silva et al. 2007) and partially by PD123,319 (=AT₂ receptor antagonist) (Ford et al. 1996), but the AT₁ antagonist (olmesartan) did not antagonize it. In most situations, Ang (1–7) and Ang II exert opposing actions, suggesting a primary role for Ang (1–7) as a counter-regulatory component for the vascular and proliferative actions of Ang II (Iwata et al. 2005; Kostenis et al. 2005). The release of nitric oxide and prostaglandins (Seyedi et al.1995; Brosnihan et al. 1996;Santos et al. 2000) might explain the lowering of IOP by Ang (1–7). Based on such and the present findings, it would seem justifiable to assume that the RAS with its different components not only regulates blood pressure but is also involved locally in the regulation of IOP and may have a role in retinal vascular diseases.

Conclusion

To the authors’ knowledge, the present findings and our previous observation of an oculohypotensive effect of Ang (1–7) constitute the first evidence that Mas receptors are expressed in eye tissue. Vasorelaxing Ang (1–7) is considered to be an endogenous ligand for the Mas receptor, which is distinct from the classical AT₁ and AT₂ receptors. The receptor density of AT₁ and AT₂ measured by autoradiography was not related to the systemic hypertension but more to the ARB medication.