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1From the Laboratory of Retinal Cell Biology and the 2Departments of Ophthalmology, 3Internal Medicine, and 4Cell Differentiation, Keio University School of Medicine, Tokyo, Japan; the 5Department of Ophthalmology, Kobe City General Hospital, Kobe, Japan; and the 6Faculty of Applied Biological Science, Gifu University, Gifu, Japan.
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METHODS. EIU was induced in Long-Evans rats by a single intraperitoneal injection of 100 µg lipopolysaccharide (LPS). Tissue localization of total prorenin, prorenin receptor, and activated prorenin in the EIU retina was examined by immunohistochemistry. To inhibit the prorenin receptor-mediated upregulation of the RAS, a decoy handle-region peptide (HRP) was intraperitoneally administered 24 hours before and immediately after the injection of LPS. Twenty-four hours after LPS injection, leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. In addition, leukocyte infiltration into the vitreous cavity and protein concentration in the anterior chamber were also measured. Retinal mRNA and protein levels of intercellular adhesion molecule (ICAM)-1, interleukin (IL)-6, and C-C chemokine ligand (CCL) 2/monocyte chemotactic protein (MCP)-1 were examined by RT-PCR and ELISA.
RESULTS. Retinal vessels in rats with EIU were strongly positive for total prorenin, prorenin receptor, and activated prorenin. Systemic treatment with HRP resulted in dose- and time-dependent inhibition of the leukocyte adhesion and infiltration and the protein leakage, all of which were increased by the induction of EIU. Retinal mRNA expression and protein levels of ICAM-1, CCL2/MCP-1 and IL-6, induced in rats with EIU, were also significantly suppressed with application of HRP.
CONCLUSIONS. These findings demonstrate for the first time that nonproteolytically activated prorenin plays a significant role in the development of ocular inflammation in the EIU model. The present study suggests the potential use of HRP, a decoy peptide binding to the prorenin receptor, as a therapeutic agent to reduce ocular inflammation.
,5 and C-C chemokine ligand (CCL) 2/monocyte chemotactic protein (MCP)-1,6 which contribute to the development of EIU, resulting in the breakdown of the blood-ocular barrier and in the infiltration of leukocytes. For the first phase of leukocyte infiltration, cell adhesion to vascular endothelium, in which adhesion molecules play major roles, is essential.7 Among various adhesion molecules, intercellular adhesion molecule (ICAM)-1 and its receptor, lymphocyte function–associated antigen (LFA)-1, are necessary for the development of EIU.7 8 9 Although EIU was originally used as a model mimicking anterior uveitis, increasing evidence shows EIU as involving inflammation in the posterior segment of the eye with recruitment of leukocytes adhering to the retinal vasculature and infiltrating the vitreous cavity.10 11 Recently, we demonstrated that angiotensin II type 1 receptor (AT1-R) blockers, widely and safely used for anti-hypertensive therapy, have an inhibitory effect on ocular inflammation in the EIU model, which is complicated by upregulated expression of AT1-R on retinal vessels.12 This finding is supported by several recent reports showing that the renin-angiotensin system (RAS), originally regarded as an important controller of systemic blood pressure, plays crucial roles in pathologic vascular conditions including inflammation and neovascularization via interaction of angiotensin II with AT1-R.13 14 15 16 The initial step for upregulation of the RAS is classically known as proteolytic activation, whereby prorenin is converted to the active (mature) form of renin by the processing enzymes to remove the prorenin prosegment, which folds into an active-site cleft of mature renin. Renin is well known to be a rate-limiting enzyme in the RAS for the cleavage of angiotensinogen to angiotensin I, which angiotensin-converting enzyme processes to angiotensin II, a final effector molecule that interacts with its cognate receptors, AT1-R and AT2-R.
In addition to the proteolytic activation of prorenin, nonproteolytic activation of prorenin, which was recently demonstrated in vitro,17 has attracted growing attention as a local upregulator of the RAS, causing organ damage. In the mechanism of nonproteolytic activation, when the prorenin binding proteins interact selectively with the handle region of the prorenin prosegment, prorenin undergoes conformational change with exposure of the active center and obtains enzymatic bioactivity of renin without cleavage of the prorenin prosegment or change in molecular weight. In vitro nonproteolytic activation of prorenin was originally observed under acidic pH or low-temperature,18 19 20 which are called acid-activation and cryo-activation, respectively. However, these experimental phenomena have never been found in vivo.
Our recent report21 indicated the importance of interaction of prorenin receptor with the handle region of the prorenin prosegment for in vivo nonproteolytic activation of prorenin (Fig. 1A) and revealed the association of nonproteolytic activation with pathogenesis in the kidney. When rats with streptozotocin-induced diabetes received a peptide with the structure of the handle region of the prorenin prosegment (handle-region peptide [HRP]; Fig. 1B ) as a decoy for the prorenin receptor, it potently suppressed the progression of diabetic nephropathy by inhibiting the nonproteolytic activation of prorenin and the subsequent upregulation of the RAS in the kidney. We have suggested the importance of this novel receptor-associated prorenin (RAP) system as an organ-specific RAS enhancer in disease, because the RAS, independently of soluble processing enzymes, is locally upregulated by nonsoluble, membrane-bound prorenin receptor in target organs.
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Preparation of Rat Prorenin HRP and Anti-HRP Antibody
Figure 1B shows the prosegment of rat prorenin. To cover the handle region (position 11–15),17 we designed a decapeptide, NH2-RILLKKMPSV-COOH, as an HRP of rat prorenin and purified it by high-pressure liquid chromatography (HPLC) on a C-18 reversed-phase column, as previously described.21 The purity and retention time of HPLC was 97.6% and 26.2 minutes, respectively. The mass of the product was 1185.7 and similar to the theoretical mass value (1186.0). The specific inhibitory action of HRP against prorenin activation was recently confirmed by using recombinant rat prorenin and COS-7-transfected cells expressing rat prorenin receptor (accession number AB188298 in the DNA Databank of Japan),21 which was originally cloned by Nguyen et al.22
An anti-rat HRP antibody was raised against a peptide, RILLKKMPSVC, conjugated with keyhole limpet hemocyanin in rabbits. HRP was used for determining the titer of the antiserum with an ABC-AP rabbit IgG kit (Vectastatin; Vector, Burlingame, CA) and as the ligand of an affinity column for purification of the antibody. High-titer antisera were obtained 6 weeks after the first immunization. The affinity gel was prepared by conjugation of an amine-coupled gel (Biogel 102; Bio-Rad, Tokyo, Japan) through a cysteine residue of antigen peptide as a ligand. The antibody was purified with the affinity column, and the concentration of the purified antibody (3.90 mg/mL) was calculated using an extinction coefficient of 1.35 at 1 mg/mL IgG and 280 nm.
Treatment with HRP
Animals were treated with 0.1-mL intraperitoneal injections of vehicle (PBS) or HRP (0.1 or 0.01 mg/kg body weight) the day before and immediately after the injection of LPS. The effects of HRP treatment on ocular inflammation were evaluated 24 hours after LPS injection.
Immunohistochemistry for Total Prorenin, Prorenin Receptor, and Activated Prorenin
Immunohistochemical experiments were performed for the rat eyes with EIU. For histopathologic evaluation, the specimen was fixed with 4% paraformaldehyde (PFA) at 4°C immediately after removal and embedded in paraffin. After 3-µm deparaffinized sections were pretreated with proteinase K, the sections were boiled in citrate buffer with microwaves to unmask antigenic sites, and endogenous biotin was blocked (Biotin Blocking System X0590; Dako, Carpinteria, CA). The sections were then immersed in 3% H2O2 in methanol, to inhibit endogenous peroxidase, and were precoated with 1% nonfat milk in PBS to block nonspecific binding. For immunohistochemical staining of total prorenin (natural and nonproteolytically activated) or prorenin receptor, the rabbit anti-rat HRP antibody (1:3200) or a goat anti-rat prorenin receptor antibody (1:100) was applied, respectively, to the sections as the primary antibody. The anti-prorenin receptor antibody was raised by using the previously established COS-7 cells producing rat prorenin receptor protein.21 The sections were incubated with a biotin-conjugated anti-rabbit IgG or biotin-conjugated anti-goat IgG as the secondary antibody. For immunohistochemical staining of activated prorenin, a goat polyclonal antibody against the active center of renin (1:1000), which cross-reacts with both nonproteolytically and proteolytically activated prorenin but not with natural prorenin,23 24 25 was applied to the sections as the primary antibody. The anti-activated prorenin antibody was kindly provided by Tadashi Inagami (Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN). The sections were incubated with a biotin-conjugated anti-goat IgG as the secondary antibody. The immunohistochemical reactions were visualized by using a Vectastain ABC Standard Kit (Vector) and 0.2 mg/mL 3,3'-diaminobenzidine tetrahydrochloride (DAB; Dojindo, Kumamoto, Japan) in 0.05 M Tris-HCl (pH 7.6) containing 0.003% H2O2. The sections were counterstained with hematoxylin. As a negative control for staining, the first antibodies were replaced with a nonimmune rabbit or goat IgG (Dako). The immunohistochemical experiments included 6 to 18 sections from two rat eyes with EIU for each antibody.
Lectin Labeling of Retinal Vasculature and Adherent Leukocytes
The leukocytes adhering to the retinal vasculature were imaged by perfusion-labeling with fluorescein-isothiocyanate (FITC)-coupled concanavalin A lectin (Con A; Vector), as described previously.26 After deep anesthesia, the chest cavity was opened and a 20-gauge perfusion cannula was introduced into the aorta. After injection of 20 mL of PBS to remove erythrocytes and nonadherent leukocytes, 20 mL of FITC-conjugated Con A lectin was perfused. Residual unbound Con A was removed with PBS perfusion. After the eyes were enucleated, the retinas were flatmounted. The flatmounts were imaged with an epifluorescence microscope (IX71; Olympus, Tokyo, Japan) and the total number of Con A–stained adherent leukocytes per retina was counted.
Aqueous Humor Analyses
Aqueous humor was collected by anterior chamber puncture with a 27-gauge needle at 0, 12, and 24 hours after LPS injection in vehicle- and HRP (0.1 or 0.01 mg/kg body weight)-treated EIU rats. Protein concentration was determined with a protein quantification kit (Dojindo), and absorbance was measured with a microplate reader (Bio-Rad).
Reverse Transcription–Polymerase Chain Reaction for ICAM-1, CCL2/MCP-1, and IL-6
Total RNA was isolated from the retina with extraction reagent (TRIzol; Invitrogen, Carlsbad, CA) and reverse-transcribed with a cDNA synthesis kit (First-Strand; GE Healthcare, Piscataway, NJ) according to the manufacturer’s protocols. PCR was performed with Taq DNA polymerase (Toyobo, Tokyo, Japan) in a thermal controller (MiniCycler; MJ Research, Watertown, MA). The primer sequences were as follows: 5'-ATG TGG CAC CAC ACC TTC TAC AAT GAG CTG CG-3' (sense) and 5'-CGT CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-3' (antisense) for ß-actin; 5'-AGC CTC AGG CCT AAG AGG AC-3' (sense) and 5'-AGG GGT CCC AGA GAG GTC TA-3' (antisense) for ICAM-1; 5'-CTG GGC CTG TTG TTC ACA GTT GC-3' (sense) and 5'-CTA CAG AAG TGC TTG AGG TGG TTG-3' (antisense) for CCL2/MCP-1; and 5'-AAA ATC TGC TCT GGT CTT CTG G-3' (sense) and 5'-GGT TTG CCG AGT AGA CCT CA-3' (antisense) for IL-6.
Enzyme-Linked Immunosorbent Assay for ICAM-1, CCL2/MCP-1, and IL-6
The animals were killed with an overdose of anesthesia, and the eyes were immediately enucleated. The retina was carefully isolated and placed into 200 µL of lysis buffer supplemented with protease inhibitors and then sonicated. The lysate was centrifuged at 15,000 rpm for 15 minutes at 4°C. The levels of ICAM-1, CCL2/MCP-1, and IL-6 in the supernatant were determined with the rat ICAM-1 kit (R&D Systems, Minneapolis, MN), the rat CCL2/MCP-1 kit (BioSource International, Camarillo, CA), and the rat IL-6 kit (R&D Systems), according to the manufacturers’ protocols. The tissue sample concentration was calculated from a standard curve and corrected for protein concentration.
Morphometric and Statistical Analyses
All results are expressed as the mean ± SD. The data were processed for statistical analyses with the Mann-Whitney test. Differences were considered to be statistically significant at P < 0.05.
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Effects of HRP on Anterior Chamber Protein Leakage
To evaluate the anti-inflammatory effect of HRP on anterior uveitis, we analyzed protein leakage into the aqueous humor. Protein concentration in the aqueous humor of the HRP-treated EIU rats was compared with vehicle-treated EIU rats (Fig. 4) . Protein concentration of the rats treated with HRP was significantly lower than that of the vehicle-treated EIU rats at 12 hours after LPS injection, and the decrease was dose dependent (Fig. 4) . At 24 hours after LPS injection, protein concentration of the rats treated with HRP (0.1 mg/kg) was significantly lower than that of the vehicle-treated EIU rats, while no significant difference was detected between the HRP (0.01 mg/kg)-treated EIU rats and vehicle-treated EIU rats (Fig. 4) .
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Discussion |
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The present study first shows the tissue localization of prorenin receptor, together with total and activated prorenin in retinal vessels (Fig. 2) . Prorenin is known to be produced in various organs including the kidney, brain, testis, ovary, and vascular endothelium. Also in the eye, prorenin was found to be present in the human surgical samples29 30 and in the rodent retina.31 32 Vitreous aspirates from patients with proliferative diabetic retinopathy contained the increased levels of prorenin.30 In the normally developing retina,32 consistent with our data from the EIU retina (Fig. 2A) and the adult retina (data not shown), prorenin is detected immunohistochemically in the retinal vessels. In contrast, prorenin receptor, recently identified and characterized,17 21 22 was shown to be produced in the eye (Fig. 2B) , whereas it had already been found in the heart, brain, placenta, liver, pancreas, and kidney.22
Furthermore, the present study shows that HRP, a decoy peptide for prorenin receptor, suppresses EIU-related ocular inflammation including leukocyte adhesion to the retinal vessels (Figs. 3A 3B) , leukocyte infiltration into the vitreous (Fig. 3C) , and protein leakage into the anterior chamber (Fig. 4) in a time- and dose-dependent manner. These findings provide the first evidence that nonproteolytic activation of prorenin in the RAS plays a significant role in ocular inflammation.
Leukocyte adhesion to the vessel walls is an important process of inflammation. When leukocytes are recruited to inflammatory sites, adhesion molecules play essential roles in the first step of inflammation. ICAM-1 and its counterreceptor ß2 (CD18) integrins (i.e., LFA-1 and Mac-1) regulate leukocyte-endothelial interaction in the pathogenesis of EIU.7 8 9 During the development of EIU, ICAM-1 is upregulated and expressed on vascular endothelial cells of the iris and the ciliary body shortly after LPS injection.8 In addition, several studies demonstrate that treatment with anti-ICAM-1 antibodies significantly inhibited the development of EIU.8 9 In the present study, retinal ICAM-1 upregulation in EIU was suppressed after treatment with HRP (Figs. 5 6A) . This is likely to result from the suppression of the RAS, activated in EIU with AT1-R upregulation,12 after HRP-induced inhibition of nonproteolytic activation of prorenin. Recent in vivo and in vitro data12 15 33 showed that angiotensin II signaling induces ICAM-1 expression via AT1-R. Collectively, the suppression of retinal inflammation observed in our study depends in part on HRP-induced inhibition of ICAM-1 via RAS downregulation. Our recent data18 showed that HRP administration results in the inhibition of nephropathy in rats with streptozotocin-induced diabetes, whereas ICAM-1 deficiency is also protective against diabetic nephropathy in db/db mice.34
Besides ICAM-1, various chemical mediators are involved in the pathogenesis of EIU. In the present study, HRP treatment led to the suppression of EIU-induced, inflammation-related molecules, including ICAM-1, IL-6, and CCL2/MCP-1. Proinflammatory effects of angiotensin II are attributable to its induction of these inflammation-related molecules, most of which are downstream products of nuclear factor (NF)-
B, a transcription factor that promotes the gene expression of various inflammatory cytokines.35 LPS-induced inflammation is mediated by the activation of NF-B.35 Indeed, ocular inflammation is suppressed by administration of an NF- B inhibitor in EIU.36 All evidence taken together, anti-inflammatory effects of HRP are likely to result from suppressed gene expression of NF-B–induced molecules. These previous findings, in accordance with our data, suggest that HRP affects not only ICAM-1-mediated leukocyte adhesion but also various inflammatory processes.
In the present study, although leukocyte adhesion was substantially suppressed with HRP (0.01 mg/kg) 24 hours after LPS injection (Fig. 3B) , little or no significant change was detected in protein leakage when the same dose of HRP was used at the same time point (Fig. 4) . Similar discrepancy between cell adhesion and infiltration and protein leakage was also noted in several EIU studies by using neutralizing antibodies against ICAM-1,8 LFA-1,8 9 E-selectin,37 P-selectin,37 and IL-10.38 Considering that prostaglandin E2, an inflammatory mediator in addition to the adhesion molecules, is operative for protein leakage39 and that combined inhibition of both L- and P-selectin suppresses protein leakage,11 37 the cell-protein discrepancy observed in the present and previous studies is most likely attributable to differential mechanisms controlling the multiple inflammatory phases.
Although our recent report12 showed that the RAS is associated with ocular inflammation in the EIU model, the present study is the first to reveal the more detailed mechanism of nonproteolytic activation of prorenin which plays a significant role in RAS-mediated ocular inflammation. Clinically, ocular inflammation such as chronic endogenous uveitis is treated mainly with topical and/or systemic application of corticosteroids. During long-term treatment with corticosteroids, however, care must be taken to guard against both ocular and systemic complications including cataract, glaucoma, diabetes, hypertension, and osteoporosis. Therefore, the establishment of additive anti-inflammatory approaches is desirable to decrease the rate and the degree of corticosteroid-induced complications. Targeting nonproteolytically activated prorenin may prove to be useful as a novel therapeutic strategy for ocular inflammation.
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Disclosure: S. Satofuka, None; A. Ichihara, None; N. Nagai, None; K. Yamashiro, None; T. Koto, None; H. Shinoda, None; K. Noda, None; Y. Ozawa, None; M. Inoue, None; K. Tsubota, None; F. Suzuki, None; Y. Oike, None; S. Ishida, 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: Susumu Ishida, Laboratory of Retinal Cell Biology, Department of Ophthalmology, Keio University School of Medicine; 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan; ishidasu{at}sc.itc.keio.ac.jp.
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