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From the Department of Physiology, The University of Melbourne, Melbourne, Victoria, Australia.
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Abstract |
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METHODS. Transgenic Ren-2 rats, which overexpress the RAS, and Sprague-Dawley (SD) rats were studied at postnatal day (P)1, P7, P14, P21, and P90. Immunohistochemistry was performed for angiotensinogen, prorenin, angiotensin II (Ang II), and the angiotensin type 1 (AT1) and 2 (AT2) receptors. Retinal active renin and prorenin were measured by radioimmunoassay, and the density of angiotensin-converting enzyme (ACE) by autoradiography. At P1 to P7, Ren-2 and SD rats were administered either the ACE inhibitor lisinopril (10 mg/kg per day, intraperitoneally [IP]) or the AT1 receptor antagonist losartan (10 mg/kg per day, IP), and vessel length and density were measured.
RESULTS. At all time points, RAS components were localized to blood vessels and cells in the ganglion cell layer. At P1, Ang II and both the AT1 and AT2 receptors were on hyaloid vessels. ACE binding increased in intensity from P1 to P90. Retinal renin was mainly activated and was 5- to 15-fold higher in Ren-2 than in SD rats. In Ren-2 rats, the growing vasculature extended farther into the retinal periphery than in SD rats and was unchanged with either lisinopril or losartan. Vascular density was increased in the periphery of Ren-2 rats compared with SD rats and was reduced with lisinopril but not with losartan.
CONCLUSIONS. In the developing rat retina, a complete RAS is mainly found in blood vessels and cells in the ganglion cell layer, where it may influence the early stages of vascularization.
Ang II exerts its actions primarily through two receptor subtypes: the Ang II type 1 (AT1) and 2 (AT2) receptors. Virtually all the biological actions of Ang II are mediated through the AT1 receptor. and including blood pressure regulation, cell growth, angiogenesis, and growth factor induction.12 13 In retinal endothelial cells, Ang II stimulates proliferation via the AT1 receptor, which involves upregulation of the potent angiogenic, vascular permeability, and endothelial cell survival factor vascular endothelial growth factor (VEGF).12 14 15 The functional role of the AT2 receptor is not fully understood, and there is evidence that it may oppose the actions of the AT1 receptor.13 In addition, the AT2 receptor has been reported to have pro-, anti-, or no angiogenic effects.13 16 17 18 High expression of this receptor subtype in fetal and neonatal tissue, with relatively low or absent levels in adult tissues19 has led to the suggestion that the AT2 receptor may be involved in the regulation of cell growth and differentiation in developing organs.13
The presence of the constituents of the RAS in the eye implies a physiological function of the system. Indeed, it is thought that Ang II contributes to the regulation of the ophthalmic circulation20 21 and to the control of aqueous humor dynamics and intraocular pressure.22 The localization of Ang II within various neuronal cell types in the retina6 10 23 has also led to the hypothesis that Ang II acts as a neuromodulator within the eye, and electrophysiological studies have suggested a functional role for the RAS in the visual system.24 25 26 Furthermore, the potent angiogenic- and growth factor–inducing properties of Ang II12 27 28 29 30 have implicated this molecule in the pathogenesis of ocular angiogenesis in experimental diabetes31 and in models of oxygen-induced retinopathy.32 33 34
The developing retina is characterized by glial migration and subsequent vascularization.35 36 37 Currently, no studies have been undertaken to examine the contribution of the RAS to angiogenesis in the developing retina. Because the standard laboratory rat displays relatively low tissue renin and angiotensin, we chose to study the transgenic m(Ren-2)27 rat (Ren-2).38 Derived from the insertion of the murine Ren-2 gene into the genome of the Sprague-Dawley (SD) rat, the Ren-2 rat displays elevated renin and Ang II in tissues except the kidney, high plasma prorenin similar to the human phenotype, and fulminant hypertension. The Ren-2 rat has facilitated the study of the extrarenal RAS in various tissues, including the retina.1 3 5 31 39 Our first objective was to evaluate the location of RAS components in the developing retina of Ren-2 and SD rats and to make comparisons to the mature eye. Second, we sought to determine whether the RAS influences the early stages of vascularization in the immature retina. This was assessed in neonatal Ren-2 and SD rats after blockade of the RAS with the ACE inhibitor lisinopril and the AT1 receptor antagonist losartan.
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Methods |
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At P1, homozygous Ren-2 rat and SD pups were randomized to the following groups: untreated control (vehicle), the ACE inhibitor lisinopril (10 mg/kg per day) and the AT1 receptor antagonist losartan (10 mg/kg per day). All agents were administered at the same time of day by intraperitoneal injection with sterile saline (pH 7.4), as the vehicle. The injection volume was 100 µL. Agents were administered from postnatal day (P)1 to P7. The doses of lisinopril and losartan were based on previous studies in oxygen-induced retinopathy, where they reduced retinal angiogenesis.32
In both studies 1 and 2, homozygous Ren-2 mothers were withdrawn from maintenance anti-hypertension therapy (lisinopril, 10 mg/kg in drinking water) 3 weeks before mating and continued without antihypertensive therapy during pregnancy and the weaning period. Mothers were allowed drinking water ad libitum. All investigations adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and to the guidelines of the University of Melbourne’s Animal Experimentation Ethics Committee.
Study 1: Characterization of the RAS in the Developing Rat Retina
On P1, P7, P14, P21, and P90, rats were killed by anesthetic overdose
(Nembutal, 60 mg/kg body weight, intraperitoneally [IP];
Boehringer-Ingelheim, North Ryde, NSW, Australia). Eyes were fixed in
Bouin’s fixative (Pathtech Diagnostics Pty. Ltd., Melbourne, Victoria,
Australia) for approximately 24 hours and then processed to paraffin,
embedded, and serially sectioned at 3 µm. It is our experience that
this method of fixation and tissue processing allows excellent
preservation of retinal morphology and antigenicity of RAS
components.1 Sections were mounted onto slides precoated
with 1% gelatin and baked overnight at 37°C. These were immersed in
clearing solution (VWR International, Darmstadt, Germany) to
remove paraffin wax, hydrated in graded ethanol, immersed in tap water,
and incubated for 10 minutes with normal goat serum (NGS; Zymed
Laboratories, South San Francisco, CA) diluted 1:10 with 0.1 M
phosphate-buffered saline (PBS) at pH 7.4. The sections were then
incubated with specific primary antibodies for 20 hours at 4°C.
Sections incubated with NGS instead of primary antibodies were used for
negative control experiments. Thorough rinsing of the sections with 0.1
M PBS (three times for 5 minutes each) was followed by incubation with
a solution of 20% hydrogen peroxide in methanol for 10 minutes and
another rinse with PBS (1 x 5 minutes). The sections were then
incubated for 30 minutes with biotinylated swine-goat-mouse-rabbit
linker antibody (Dako Corp., Carpinteria, CA) diluted 1:90 in 0.1 M PBS
for 1 hour, and then rinsed with 0.1 M PBS (1 x 5 minutes).
Sections were then incubated for 45 minutes with avidin-biotin
peroxidase complex (equal volumes of each component diluted 1:200 in
0.1 M PBS; Vectastain ABC kit; Vector Laboratories, Burlingame, CA) and
liquid diaminobenzidine substrate-chromogen solution (Dako Corp.) was
used as a chromogen for immunoperoxidase staining. The sections were
then rinsed in tap water, stained in Mayer’s hematoxylin,
differentiated in Scott’s tap water, dehydrated in alcohol, cleared,
(Histolene; Fronine Pty. Ltd., Riverstone, NSW, Australia) and mounted
(DPX medium; BDH Laboratories, Poole, UK). Six to eight randomly chosen
sections per eye and six to eight eyes from each animal group were
used.
The primary antibodies were angiotensinogen, prorenin, Ang II, and the AT1 and AT2 receptors. The polyclonal rabbit anti-rat angiotensinogen antibody40 was diluted 1:1000 with 0.1 M PBS. The angiotensinogen antibody was a gift from Conrad Sernia (Department of Physiology and Pharmacology, University of Queensland, Australia). The monoclonal rabbit anti-mouse prorenin antibody is targeted at 13 amino acids in the prosequence (PSVREILEERGVD). The prorenin antibody was used at dilutions of 1:1500 to 1:2000 in 0.1 M PBS and was a gift from Geoff Tregear (Howard Florey Institute, Melbourne, Australia). The polyclonal guinea pig anti-human Ang II antibody (Peninsula Laboratories Inc., Belmont, CA), was used at dilutions of 1:750 to 1:1500 in 0.1 M PBS. The polyclonal antibody to the AT1 receptor (sc 1173) and to the AT2 receptor (sc 9040; Santa Cruz Biotechnology, Santa Cruz, CA), were diluted 1:200 in 0.1 M PBS. Confirmation of specific antibody labeling was achieved in sections of rat liver (angiotensinogen) and kidney and ovary (prorenin, Ang II and AT1 and AT2 receptors).
A separate group of Ren-2 and SD rats were killed by anesthetic overdose at P1, P7, P14, P21, and P90. Enucleated eyes were embedded in optimal cutting temperature compound (Cryomatrix; Sakura Finetechnical Co., Ltd., Tokyo, Japan), immersed in isopentane, snap frozen in liquid nitrogen and stored at –80°C. Sections were cut at 20 µm on a cryostat at –20°C, thaw mounted onto glass slides precoated with 3-aminopropyltriethoxysilane, dehydrated under reduced pressure at 4°C overnight, and stored at –80°C. A tyrosol derivative of the ACE inhibitor lisinopril, 351A was radioiodinated with 125I, using chloramine T and purified by SP-Sephadex C25 chromatography. The binding properties and use of this radioligand have been extensively described.1 41 Five randomly chosen sections per eye and six to eight eyes from each animal group were used.41 Brain and kidney sections were used as controls, as ACE has been localized in these tissues. Sections were brought to room temperature and preincubated for 15 minutes in sodium phosphate buffer (150 mM NaCl, 10 mM Na2HPO4) containing 0.2% bovine serum albumin (BSA, pH 7.4). They were then transferred to fresh buffer containing 0.3 Ci/mL of [125I]351A for 1 hour at room temperature. For the determination of nonspecific binding, parallel incubations were performed in the presence of 1 mM EDTA (BDH Laboratory Supplies). After incubation, the sections were transferred through four successive 1-minute washes in ice-cold fresh buffer (excluding BSA) and dried under a stream of cold air. Dry slides were placed in cassettes, including a set of radioactivity standards, and exposed to x-ray film (Agfa-Scopix CR3; Agfa-Gevaert, Melbourne, Australia) for 3 days, and the films were then developed (Department of Radiology, Austin Hospital, Heidelberg, Victoria, Australia).
Autoradiographs for radioligand binding were quantitated by computerized image analysis (Analytical Imaging Station; Imaging Research, Ontario, Canada). Developed films were placed on a uniformly illuminating fluorescent light box and images captured with a digital camera (Fujix HC-2000; Fuji, Tokyo, Japan). Radioactive standards were fitted to calibration curves, and the optical density of each pixel of digitized image converted into disintegration per minute of I125 per square millimeter of tissue. Nonspecific binding was negligible. Sections of rat kidney and brain served as the positive control.
A separate group of Ren-2 and SD rats were killed by anesthetic overdose at P1, P7, P14, P21, and P90. Total renin (prorenin plus active renin) and active renin were estimated in eyes by an established technique.1 5 31 32 Prorenin is derived as total renin minus prorenin. Rats were anesthetized (60 mg/kg body weight IP, Nembutal; Boehringer-Ingelheim), eyes enucleated, and the lenses and vitreous removed. To estimate total renin, right eyes were snap frozen in 0.1 M PBS at pH 7.4. To estimate active renin, left eyes were snap frozen in a 30% protease inhibitor solution containing 25 mM N-ethyl maleimide, 20 mM EDTA, and 100 mM benzamide. All samples were stored at –20°C and later thawed, homogenized, and refrozen twice before assay by an enzyme kinetic method with hog renin (National Standards Laboratory, London, UK) as the reference standard and 24-hour nephrectomized rat plasma as the angiotensinogen substrate. Duplicate samples were incubated for 1 hour at 37°C (pH 7.4), and the renin present in the samples was estimated by immunoassay of generated Ang I referenced against the amount of Ang I generated by 2 x 10–6 Goldblatt units (GU) of hog renin. Total renin was assayed by using tissue incubated without inhibitors after activation of prorenin by trypsin treatment (2 mg/mL for 10 minutes on ice).
Study 2: Developmental Retinal Angiogenesis and RAS Blockade
At P7, Ren-2 and SD rats were killed by anesthetic overdose and retinas
dissected and fixed for 2 hours in 4% paraformaldehyde (BDH Laboratory
Supplies) diluted in 0.1 M PBS. The retinas were then permeabilized
overnight in 0.1 M PBS containing 0.5% Triton X-100 (Sigma-Aldrich,
St. Louis, MO) and 5% NGS. These were subsequently incubated with the
endothelial cell marker FITC Griffonia
(Bandeiraea) simplicifolia lectin B4
(Vector Laboratories, Burlingame, CA), diluted 1:80 in 0.1 M PBS
containing 0.5% Triton X-100 (PBS/Triton X-100 solution), for 6 hours
at room temperature. Retinas were washed for 15 minutes with PBS/Triton
X-100 solution and stored overnight on a circular shaker at 4°C in
clean PBS/Triton X-100 solution. Retinas were subsequently washed again
(four tomes for 15 minutes each) with PBS/Triton X-100 solution at room
temperature and then incubated for 3 hours at room temperature with
FITC-streptavidin (Dako Corp.) diluted 1:100 in PBS/Triton X-100
solution. This process was followed by six 15-minute washes with
PBS/Triton X-100. Retinas were then flat mounted on clean glass slides
with fluorescent mounting medium (Dako Corp.) and stored flat in the
dark at 4°C for 1 week.
Low-power images of each quadrant of the flatmounted retinas, encompassing the area from the optic disc to the peripheral edge of the section, were captured with a digital camera (Spot; SciTech Pty. Ltd., Preston, Victoria, Australia) attached to a reflected fluorescence microscope (BX52; Olympus, Tokyo, Japan) with a narrow-band excitation filter for detection of FITC fluorescence. Retinal vascular density was evaluated with appropriately calibrated computerized image analysis (Analytical Imaging Station; Imaging Research), a technique based on methodology previously described.42 Vascular density in each region (central, middle, and peripheral retina) was determined by measuring the proportional area of fluorescently labeled vessels in two 200 x 200-µm sampling fields. Sampling fields were chosen to ensure they were not bisected by a major artery or vein. From the optic disc, the central retina was defined as a distance of up to 1.25 mm, the middle retina between 1.25 and 2.5 mm, and the peripheral retina beyond 2.5 mm. Six to eight retinas were analyzed per animal group.
Low-power images used for the quantitation of retinal vessel density were also used in the quantitation of vessel length, which was evaluated with appropriately calibrated computerized image analysis (Analytical Imaging Station; Imaging Research), based on methodology previously described.43 Briefly, vessel length was measured from the optic disc to the edge of the vascular front in all four retinal quadrants, and an average value was taken for each retina. Six to eight retinas were analyzed per animal group.
Statistical Analysis
Statistical analysis was performed using one-way ANOVA with post
hoc analysis by the Student-Newman Keuls test for multiple comparison
of individual mean values. Data for the Ang II receptor autoradiography
were log transformed.
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Results |
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Angiotensinogen, Prorenin, Ang II, and AT1 and AT2 Receptor Localization.
Angiotensinogen protein labeling was localized to the cytoplasm of
cells in the ganglion cell layer (GCL) and to blood vessels in the
region of the GCL and inner limiting membrane (ILM) at all time points
in both Ren-2 (Fig. 1) and SD rats (Fig. 1) . Only in Ren-2 rats was angiotensinogen also
localized to the somas of cells in the middle of the inner nuclear
layer (INL) at P14, P21, and P90 (Figs. 1C 1D 1E) .
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Discussion |
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Gene expression studies have identified that a tissue-based RAS exists in the adult eye. Renin, angiotensinogen, and ACE mRNA have been detected in retinal pigment epithelium/choroid and whole neural retina samples from human eyes.8 In situ hybridization and immunohistochemical studies have identified the retinal cell types in which RAS components are located. In the inner retina of the adult rat, mouse, rabbits, and humans, the RAS is found in blood vessels, neurons, and glia.6 7 9 10 In the present study, RAS components were located in similar sites from as early as P1 and persisted through retinal development and into adulthood. The finding of RAS components in blood vessels of the immature rat retina and Ang II and AT1 and AT2 receptor protein in hyaloid vessels (a transiently existing network of capillaries that nourish the developing lens and retina) suggests a role for the RAS in vascularization of the developing retina. With regard to ACE, previous studies have reported immunoreactivity in retinal vessels and in blood vessels in other organs.46 In the present study, autoradiographic analysis showed retinal ACE to increase with retinal maturation, with highest levels in the adult at P90. In general, Ren-2 rats had slightly lower levels of retinal ACE than did SD rats. This was most notable at P7 and P90. The reason for this strain difference is probably the overexpression of the retinal RAS in Ren-2 rats, which has the effect of suppressing tissue ACE levels. Our previous studies support this finding, as adult Ren-2 rats have reduced levels of plasma ACE compared with SD rats.47 It should be noted that in several species, ACE increases over the perinatal period in lung, kidneys, heart, and aorta.46 48 49 The reason for the increase in ACE with tissue development is not precisely known, but could relate to the extent of vascularization. For instance, it has been suggested that the postnatal increase in pulmonary ACE expression in lambs may partly reflect the increase in capillary surface area associated with growth.50
In the present study, RAS components were localized to cells in the GCL, which could represent ganglion and displaced amacrine cells.51 RAS components also exist in macroglial Müller cells.6 Together, these findings suggests that the angiotensin produced in these cells exerts a paracrine effect on the neighboring vasculature. Indeed, neurons and glia in the retina are structurally aligned with the retinal vasculature,52 and contribute to the epiretinal membranes that form in diabetic retinopathy.53 Alternatively, the retinal RAS may influence neuronal activity (reviewed in Ref. 54 ). Of interest is that blockade of RAS modulates the a- and b-waves of the electroretinogram.25 ACE may also have a role in neuronal development in the retina. In chicken retina that lack blood vessels, ACE activity and gene expression increase in the developing embryo retina and peak transiently postpartum.55 56 This developmental upregulation of ACE coincides with the period of synaptogenesis in the chicken retina, leading to the suggestion that ACE may be involved in the fine tuning of the neuronal retinal network and/or the metabolism of neuropeptides, such as substance P.
The role of Ang II in the developing retina is unknown. The present study is the first to examine whether the RAS participates in the development of the rat retinal vasculature during the postnatal period. The transgenic Ren-2 rat has been extensively used by our laboratory to examine the location of RAS in tissues such as ovary, thymus, adrenal gland, and prostate and to determine its possible role in disease at these sites.1 3 5 39 In particular, when diabetes is induced in the Ren-2 rat it causes upregulation of the RAS in specific cell types in the kidney and has pathologic effects, including severe glomerulosclerosis and tubulointerstitial disease, and in the eye, endothelial cell proliferation in the retina and iris.31 57 58 In the present study, enzyme renin, the rate-limiting enzyme in the RAS cascade responsible for the liberation of Ang I from angiotensinogen, increased with retinal development and was mostly present in an activated form. Notably, the level of renin and prorenin was approximately 5 to 15 times higher in the eyes of transgenic Ren-2 rats from P1 to P90 than in age-matched SD rats. Indeed, very low levels of prorenin were found in the retinas of SD rats, so that at P7, P21, and P90 prorenin levels were below the detection level of the enzyme kinetic assay, a finding consistent with results showing tissue prorenin to be extremely low in SD rats.1 5 The overexpression of renin in the developing retina of Ren-2 rats is associated with more extensive vascularization. In the normal rat, the retinal vasculature develops between P0 and P18 and extends from the optic disc to the outermost edge of the peripheral retina.59 In transgenic Ren-2 rats at P7, the growing edge of the vasculature extended farther, to the retinal periphery, and was denser in the periphery than in SD rats, indicating that upregulation of the retinal RAS promotes retinal vascular development.
Blockade of the RAS is commonly used to determine the contribution of Ang II to disease, including angiogenesis.32 33 In neonates with oxygen-induced retinopathy, ACE inhibition reduces pathologic retinal angiogenesis in both Ren-2 and SD rats when administered from P11 to P18.32 In the present study, ACE inhibition administered from P1 to P7 had an anti-angiogenic effect in Ren-2 rats, reducing vascular density in the peripheral retina, although not affecting the length of the growing vasculature from the optic disc to the periphery. The reasons for the lack of an antiangiogenic response with ACE inhibition in the developing retina of SD rats is not clear but could be due to a number of factors. SD rat tissues have been reported to have a reduced RAS compared with Ren-2 rats1 5 and may be less sensitive to RAS blockade than Ren-2 rats. For instance, in Ren-2 rats, very low-dose RAS blockade normalizes systolic blood pressure and reduces fibrotic and angiogenic disease in both normal and diabetic tissues.60 61 Therefore, although the dose of the ACE inhibitor lisinopril used in the present study reduced pathologic angiogenesis in SD rats with oxygen-induced retinopathy, a higher dose may be needed to influence vasculogenesis in the growing SD rat retina.
In the present study, AT1 receptor blockade had no effect on vessel length or density in the retinas of either Ren-2 or SD rats when administered from P1 to P7, perhaps because of the higher abundance of AT2 than AT1 receptors in this period, particularly at P1.7 The contribution of the AT2 receptor to the early stages of retinal angiogenesis is difficult to assess because of the lack of AT2 receptor antagonists that do not necessitate continuous administration. Previous in vivo studies using the AT2 receptor antagonist, PD123319, have shown that continuous administration with miniosmotic pumps is necessary to ligate the AT2 receptor.62 In studies such as the present one, it is not technically feasible to insert miniosmotic pumps into P1 rats. Nevertheless, an antiangiogenic effect of AT2 receptor blockade has been reported in slightly older rats with oxygen-induced retinopathy, and the AT2 receptor is associated with organ development being highly expressed in fetal and developing tissues and less abundant in the adult.19
In summary, this study demonstrates that a retinal RAS is present in the vasculature and neurons of the inner retina of the rat from as early as P1. Overexpression of the RAS in transgenic Ren-2 rats is associated with augmented vascularization in the developing retina which can be reduced with ACE inhibition. AT1 receptor antagonism does not influence developmental angiogenesis in the rat retina, which may be due to the predominance of the AT2 receptor and its reported role in organogenesis. Further studies are needed to elucidate the contribution of the RAS and its receptors to angiogenesis and neuromodulation both in development and disease.
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Acknowledgements |
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Footnotes |
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Submitted for publication July 27, 2004; revised September 26 and November 9, 2004; accepted November 12, 2004.
Disclosure: S. Sarlos, None; J.L. Wilkinson-Berka, None
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Jennifer L. Wilkinson-Berka, Department of Physiology, University of Melbourne, Grattan Street, Parkville, Victoria, Australia 3010; jlaberka{at}unimelb.edu.au
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) and in the OLM (
SD rat; 
Ren-2 rat. *P < 0.05 compared with all SD
groups; #P < 0.05 between Ren-2 and SD at a given time
point; 
P < 0.05 compared with all other time points
for the Ren-2 rat; and 
P < 0.05 compared with all
Ren-2 groups.
Ren-2 rat.
*P < 0.01 comparing active renin between Ren-2 and SD
rats; #P < 0.05 comparing active renin with that in
Ren-2 rats at P1, P7, and P14. 
