The expression of Mas-receptor of the renin–angiotensin system in the human eye

Purpose The local renin–angiotensin system has been held to be expressed in many organs, including the eye. It has an important role in the regulation of local fluid homeostasis, cell proliferation, fibrosis, and vascular tone. Mas-receptor (Mas-R) is a potential receptor acting mainly opposite to the well-known angiotensin II receptor type 1. The aim of this study was to determine if Mas-R is expressed in the human eye. Methods Seven enucleated human eyes were used in immunohistochemical detection of Mas-R and its endogenous ligand angiotensin (1–7) [Ang(1–7)]. Both light microscopy and immunofluorescent detection methods were used. A human kidney preparation sample was used as control. Results The Mas-R was found to have nuclear localization, and localized in the retinal nuclear layers and in the structures of the anterior segment of the eye. A cytoplasmic immunostaining pattern of Ang(1–7) was found in the inner and outer nuclear and plexiform layers of the retina and in the ciliary body. Conclusion To the best of our knowledge, this is the first report showing Mas-R expression in the human eye. Its localization suggests that it may have a role in physiological and pathological processes in the anterior part of the eye and in the retina.


Introduction
The systemic renin-angiotensin system (RAS) regulates body fluid balance, blood pressure, and many cardiovascular responses via endocrine mechanisms. Vasoconstrictive angiotensin II (Ang II) has a key role in RAS by acting mainly via the angiotensin type 1 receptor (AT1-R) [1,2]. In addition to the classical AT1-R and angiotensin type 2 (AT2-R) receptors, another angiotensin receptor type, a Mas-receptor (Mas-R), has been identified [3,4]. Many of the Mas-R effects are opposite to those of AT1-R. Both have important but opposite roles in the regulation of local fluid homeostasis, cell proliferation, fibrosis, and vasodilatation. Mas-R is activated by an endogenous heptapeptide, angiotensin 1-7 [Ang (1-7)], which in turn is a degradation product of octapeptide Ang II (see Fig. 1 illustrating the RAS cascade). In addition to the systemic RAS, a local tissue RAS has been identified in which long-term changes are regulated by autocrine and paracrine mechanisms [5]. Local RAS has been widely described in the heart, kidney, and adrenal tissue, but also in other organs such as the brain, reproductive system, intestine [5][6][7], and the eye [8][9][10][11][12][13][14]. The present study was designed to investigate localization of Mas-R and Ang (1)(2)(3)(4)(5)(6)(7) in the human eye using immunohistochemistry.

Materials and methods
Eyes and fine-needle samples of the kidney The study was approved by the local ethical committee (Regional Ethics Committee of the Expert Responsibility Area of Tampere University Hospital, R10155). Seven human eyes were used for detection of Mas-R and its endogenous ligand Ang(1-7). The eyes had earlier been enucleated due to ocular malignancies (all melanomas) at the Tampere University Hospital Eye Center, Tampere, Finland, and were stored in paraffin blocks after removal. As control tissue we used human kidney fine-needle samples originally taken at the Tampere University Hospital for other purposes. The kidney samples were stored in paraffin as well.
Light microscopy

Negative controls
The sections were stained both by omitting primary and by omitting secondary antibody. In addition, the pre-adsorption

Expression of Mas-receptors
Mas-R is well known to be expressed in the kidney, adrenals, and vascular system cells [4][5][6] . Therefore, we chose human kidney tissue as positive control for the antibody recognizing Mas-R. In the kidney tissue, Mas-R was mainly expressed in the tubular and glomerular cells and vascular endothelium (Fig. 2).
Next, we immunostained human eye sections using the same antibody. Mas-R was widely expressed in the eye structures. In the retina, Mas-R was found in the nuclear layers as well as in the retinal pigment epithelium and choroid (Fig. 3). In ciliary body, Mas-R was expressed by non-pigmented cells and the ciliary epithelium (Fig. 4). Mas-R immunoreactivity was also observed in the trabecular meshwork and in the wall of Schlemm's canal. In the human cornea, the majority of the basal and superobasal epithelial cells were immunopositive for Mas-R (Fig. 5).
To verify subcellular localization of Mas-R, we used double immunofluorescent staining of sections with antibody directed against Emerin, a nuclear envelope protein. In corneal, ciliary body, retinal, and choroid cells, Mas-R immunoreactivity was exclusively localized in the nucleus of cells (Fig. 6).

Expression of Ang(1-7)
Ang(1-7) expression was first verified in the human kidney tissue. Thereafter, Ang(1-7) expression was identified in the human ocular sections. Similarly to Mas-R immunoreactivity, Ang(1-7) was widely expressed in the ocular tissue. We found Ang(1-7) immunoreactivity in the inner and outer nuclear and plexiform layers of retina, choroid, non-pigmented, and pigmented epithelial cells of the ciliary body (Fig. 7). In contrast to Mas-R immunoreactivity, Ang(1-7) was expressed in the cytoplasm of cells and in some cells colocalized with neuronal marker NeuN, glial cell marker GFAP and endothelial cell marker CD31 (Fig. 8).

Discussion
In this study, we describe Mas-R and its endogenous ligand Ang(1-7) immunoreactivity and cellular localization in human ocular tissue. We found that Mas-R is widely expressed in the eye, with primarily nuclear localization in cells. Ang(1-7) was also found in different structures of the eye, but with cytoplasmic localization in cells. Previously, our research group has described Mas-R expression in the rat eye using the RT-PCR method [15].
Mas-R was first discovered by Young et al. (1986) as protooncogene almost three decades ago [3]. Later, the same group reported high Mas-R levels in the rat central nervous system [16]. Two decades ago, Kitaoka and co-workers (1994) demonstrated Mas-R expression in the eyes of rhesus macaque using in-situ hybridization and epi-polarization microscopy [17]. The authors concluded that Mas-R could be used as a specific marker for retinal pigment epithelium cells [17]. However, the functional significance of Mas-R and its putative ligand Ang(1-7) expression in the ocular structures remains unknown. Mas-R is an important member of the local tissue RAS, acting mainly opposite to the well-known AT1-R.
Mas-R participates in cell proliferation and antifibrosis as well as vasodilatation and local fluid volume homeostasis. In fact, the potentials of Mas-R ligands, e.g. Ang(1-7) and active enzymes such as ACE2 in degrading vasoconstrictive Ang II to vasodilatory peptides constitute a present focus of cardiovascular drug development [18][19][20]. Most components of the local RAS have already been widely identified in eye structures [10,21]. However, further studies are required to identify the specific physiologic role of local RAS in the human eye and possible involvement in such pathological conditions as diabetic retinopathy, age-related macular degeneration, and glaucoma.
In a previous in-vivo study, we found that intraocular administration of Mas-R agonist Ang(1-7) lowers intraocular pressure (IOP) in normotensive rabbit eyes without influence on aqueous humor outflow facility [21]. In human studies, orally administered ACE inhibitor (Captopril) and AT1-R antagonist (Losartan) reduced IOP in both ocular normotensive and glaucomatous subjects [22,23]. Oculohypotensive effects of ACE inhibitor Perindopril have also been reported in both acute and chronic experimental models of glaucoma [24], as recently also the anti-glaucomatous effects of the activation of intrinsic angiotensin-converting enzyme 2 in an experimental glaucoma [9]. Our data from human eyes indicate that Mas-R is localized in the human nonpigmented epithelial cells of the ciliary body as well as in the cells of trabecular meshwork. The latter suggests that the aqueous humor production and outflow may be influenced by Mas-R and its ligand Ang(1-7). However, further studies are needed to confirm that.
To conclude, expression of RAS components and their cellular localization suggest their involvement in controlling aqueous humour dynamics and intraocular pressure. Furthermore, our descriptive data suggest that RAS may influence pathophysiology of ocular diseases affecting ocular blood flow and vasculature such as diabetic retinopathy. However, further studies are needed to understand the functional significance of the local RAS in the eye.