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B Pathway1From the Laboratory of Retinal Cell Biology, the 2Department of Ophthalmology, and the 4Laboratory of Vascular Biology and Metabolism, Keio University School of Medicine, Tokyo, Japan; the 5Department of Ophthalmology, Kobe City General Hospital, Kobe, Japan; and the 6Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Yokohama, Japan.
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Abstract |
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B pathway with diabetes-induced retinal inflammation.
METHODS. Six weeks after induction of diabetes, C57BL/6 mice were treated with the angiotensin II type 1 receptor (AT1-R) blocker (ARB) telmisartan or valsartan, the AT2-R blocker PD123319, or the NF-B inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) daily for 1 week. Retinal mRNA and protein levels of the RAS components were examined by RT-PCR and Western blot, respectively. Leukocyte adhesion to the retinal vasculature was evaluated with a concanavalin A lectin perfusion-labeling technique. Retinal expression levels of intercellular adhesion molecule (ICAM)-1 and vascular endothelial growth factor (VEGF) were examined by RT-PCR and ELISA. ARB or DHMEQ was applied to murine capillary endothelial (b-End3) cells stimulated with a high concentration of glucose to analyze nuclear translocation of NF-B via immunohistochemistry for p65 and mRNA and protein levels of ICAM-1 and monocyte chemotactic protein (MCP)-1.
RESULTS. Induction of diabetes led to a significant increase in retinal expression and production of the RAS components including angiotensin II, AT1-R, and AT2-R. Retinal adherent leukocytes were significantly suppressed by AT1-R, but not by AT2-R, blockade. Administration of the ARB, but not of PD123319, inhibited diabetes-induced retinal expression of ICAM-1 and VEGF. DHMEQ also suppressed these cellular and molecular inflammatory parameters in the diabetic retina to the levels obtained with ARB treatment. In vitro, glucose-induced nuclear translocation of NF-B p65 and upregulation of ICAM-1 and MCP-1 were significantly suppressed by application of the ARB. The in vivo treatment with the ARB, as well as DHMEQ, attenuated the diabetes-induced retinal expression of angiotensin II and AT1-R, per se.
CONCLUSIONS. The present data revealed significant a contribution of the AT1-R/NF-B pathway to diabetes-induced retinal inflammation, providing a mechanistic reason for targeting AT1-R or NF-B in the treatment of diabetic retinopathy.
The renin–angiotensin system (RAS) plays an important role in the regulation of systemic blood pressure. Angiotensin II, the final product of the system working as a constrictor of blood vessels, has two cognate receptors, angiotensin II type 1 receptor (AT1-R) and AT2-R.13 Because the major pathogenic signaling of angiotensin II is mediated by AT1-R, AT1-R blockers (ARBs) are widely used in patients with hypertension and cardiovascular diseases. Recently, various functions of the RAS have been pointed out, including angiogenesis14 15 and inflammation.16 We have shown the inhibitory effect of ARBs on several retinal disorders mediated by ICAM-1, including ischemia-induced retinal neovascularization,17 endotoxin-induced retinal inflammation,18 and choroidal neovascularization.19
Nuclear factor (NF)-B is a transcription factor activated by various stimuli such as bacterial endotoxins, inflammatory cytokines, hypoxia, and hyperglycemia, and it plays a critical role in the regulation of gene expression of inflammation-related molecules including adhesion molecules, chemokines, and cytokines.20 AT1-R downstream signaling is known to lead to the activation of NF-B.21 In alloxan-induced diabetic rats, NF-B activation was shown to be an early retinal event, possibly linked to the development of diabetic retinopathy.22
Recent clinical and experimental studies have indicated the association of the RAS with diabetic retinopathy. Clinically, angiotensin-converting enzyme (ACE) inhibition results in significant suppression of the progression of retinopathy in normotensive subjects with type 1 diabetes.23 However, molecular and cellular mechanisms by which the RAS plays a role in the pathogenesis of diabetic retinopathy remain to be elucidated, although recent in vivo experiments have demonstrated that inhibition of the RAS leads to significant amelioration of vascular hyperpermeability24 and blood flow25 in the diabetic retina. Herein, we report the in vivo and in vitro suppression of diabetes-induced retinal inflammation and glucose-stimulated endothelial reaction by blocking AT1-R or its downstream mediator NF-B, together with underlying molecular and cellular mechanisms.
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Blockade of AT1-R, AT2-R, and NF-B
Six weeks after induction of diabetes, animals were treated with an ARB (telmisartan or valsartan), an AT2-R blocker (PD123319; Sigma-Aldrich), an NF-B inhibitor (DHMEQ; dehydroxymethylepoxyquinomicin), or vehicle (0.25% dimethyl sulfoxide [DMSO] in phosphate-buffered saline [PBS]) daily for 1 week. ARBs and DHMEQ were injected into mice intraperitoneally. PD123319 was administered by subcutaneous osmotic minipump (Alzet model 2001; Alza, Palo Alto, CA), as described previously.26 Telmisartan and valsartan were kind gifts from Boehringer Ingelheim (Ingelheim, Germany) and Novartis Pharma (Basel, Switzerland), respectively. DHMEQ is a novel NF-B inhibitor, based on the structure of epoxyquinomicin C, which was originally isolated from Amycolatopsis.27 DHMEQ has been shown to inhibit nuclear translocation of NF-B without affecting phosphorylation and degradation of I-B
. Optically active (–)-DHMEQ28 was used throughout the experiments. Mice received telmisartan at a dose of 0.5 or 5 mg/kg BW, valsartan at 1 or 10 mg/kg BW, PD123319 at 1 or 10 mg/kg BW, or DHMEQ at 1 or 5 mg/kg BW. Systolic blood pressure was assessed by a computerized, noninvasive tail cuff system (MK-2000; Muromachi Kikai, Tokyo, Japan) after each 1-week treatment.
Quantification of Retinal Adherent Leukocytes
The retinal vasculature and adherent leukocytes were imaged by perfusion-labeling with fluorescein-isothiocyanate (FITC)-coupled concanavalin A lectin (Con A) (Vector, Burlingame, CA), as described previously.7 With the mouse under deep anesthesia, the chest cavity was opened and a 27-gauge cannula was introduced into the left ventricle. After injection of 2 mL of PBS to remove erythrocytes and nonadherent leukocytes, 2 mL of FITC-conjugated Con A was perfused. 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 determined.
Western Blot Analyses for AT1-R, AT2-R, and Angiotensin II
Animals were killed with an overdose of anesthesia, and the eyes were immediately enucleated. The retina was carefully isolated and placed into 100 µL of lysis buffer (0.02 M HEPES, 10% glycerol, 10 mM Na4P2O7, 100 µM Na3VO4, 1% Triton, 100 mM NaF, 4 mM EDTA [pH 8.0]) supplemented with protease inhibitors (2 mg/L aprotinin, 100 µM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 2.5 µM pepstatin A) and sonicated. The lysate was centrifuged and the supernatant was collected. Each sample containing 30 µg of total protein was separated by SDS-PAGE (sodium dodecyl sulfate–polyacrylamide gel electrophoresis), and electroblotted to PVDF (polyvinylidene fluoride) membranes (ATTO, Tokyo, Japan). After nonspecific binding was blocked with 5% skim milk, the membranes were incubated with a rabbit polyclonal antibody against angiotensin II (1:200), AT1-R (1:100), or AT2-R (1:300; Santa Cruz Biotechnology, Santa Cruz, CA) or an anti--tubulin antibody (1:2000; Sigma-Aldrich) at 4°C overnight, followed by incubation with a horseradish peroxidase–conjugated goat antibody against rabbit IgG (1:5000; BioSource, Camarillo, CA). The signals were visualized with chemiluminescence (ECL kit; GE Healthcare, Buckinghamshire, UK) according to the manufacturer's protocol.
RT-PCR Analyses for ICAM-1, VEGF, MCP-1, AT1-R, AT2-R, and Angiotensinogen
Total RNA was isolated from the retina using an extraction reagent (TRIzol; Invitrogen, Carlsbad, CA) and reverse-transcribed with a cDNA synthesis kit (First-Strand; GE Healthcare). PCR was performed with Taq DNA polymerase (Takara Bio, Ohtsu, Japan) in a thermal controller (Gene Amp PCR system; Applied Biosystems, Foster, CA). The primer sequences and the expected size of amplified cDNA fragments are indicated in Table 1 .
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In Vitro Assays
Murine brain-derived capillary endothelial cells (b-End3) were cultured with DMEM (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) and 5.5 mM D-glucose at 37°C in a 95% air-5% CO2 atmosphere. After the cells achieved confluence, they were incubated with the same medium containing normal (5.5 mM) or high (25 mM) concentration of D-glucose alone, high glucose plus telmisartan (3 µM), high glucose plus valsartan (3 µM), or high glucose plus DHMEQ (10 µg/mL) for 12 hours. To exclude bias from the effects of hyperosmolarity, the cells were incubated in a medium containing 5.5 mM D-glucose supplemented with 19.5 mM mannitol. Total cellular RNA from b-End3 cells was isolated and PCR was performed to evaluate mRNA expression of ICAM-1 and MCP-1. For protein analyses, supernatant and cell lysates were collected after a 24-hour incubation, and then the concentrations of MCP-1 in the supernatant and ICAM-1 in the cell lysates were measured by the ELISA kits (R&D Systems).
Immunocytochemistry for NF-B p65
The b-End3 cells were incubated with the culture medium containing a normal (5.5 mM) or high (25 mM) concentration of D-glucose alone, high glucose plus telmisartan (3 µM), high glucose plus valsartan (3 µM), or high glucose plus DHMEQ (10 µg/mL) for 24 hours. After incubation, cells were fixed in methanol and washed with PBS. Nonspecific binding was blocked by incubating the cells for 1 hour in PBS containing 3% normal goat serum and 0.4% Triton. These cells were then incubated with a rabbit polyclonal antibody against human NF-B p65 (1:100, Santa Cruz Biotechnology) overnight at 4°C. Cells were subsequently washed and incubated with an avidin-Alexa 488–tagged secondary antibody (1:200; Invitrogen-Molecular Probes, Eugene, OR) for 40 minutes at room temperature. For nuclear staining, the cells were treated with TOTO-3 (1:500; Invitrogen-Molecular Probes). After two washes, the cells were viewed with the epifluorescence microscope (Olympus).
Morphometric and Statistical Analyses
All results are expressed as mean ± SD. The values were processed for statistical analyses (Mann-Whitney test), and differences were considered statistically significant at P < 0.05.
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B InhibitionB inhibition with DHMEQ significantly decreased adherent leukocytes in a dose-dependent fashion (Fig. 4A) . The mRNA expression (Fig. 4B) and protein levels of ICAM-1 (Fig. 4C) and VEGF (Fig. 4D) , upregulated by induction of diabetes, were significantly suppressed by systemic application of DHMEQ at a dose of 5 mg/kg. The NF-B inhibition with DHMEQ showed equivalently (P > 0.05) suppressive effects on these diabetes-induced retinal inflammatory parameters compared with the ARB treatment (Figs. 2F 3B 3C) .
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B Activation and Inflammatory Molecules by AT1-R BlockadeB p65 in b-End3 cells, enhanced by high glucose, was significantly suppressed by application of telmisartan or valsartan to the level seen in NF-B inhibition with DHMEQ (Figs. 5A 5B) . The mRNA expression of ICAM-1 and MCP-1 in b-End3 cells, induced by the 12-hour exposure to high glucose, was substantially suppressed by treatment with telmisartan, valsartan, or DHMEQ (Fig. 5C) . AT1-R blockade with telmisartan or valsartan, as well as NF-B inhibition with DHMEQ, significantly reduced protein levels of ICAM-1 (Fig. 5D) and MCP-1 (Fig. 5E) upregulated by high glucose stimulation.
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B inhibition on the angiotensin II-AT1-R ligand-receptor system in the diabetic retina. AT1-R blockade by telmisartan (5 mg/kg) or NF-B inhibition with DHMEQ (5 mg/kg) substantially reduced retinal mRNA expression of angiotensinogen and AT1-R upregulated by inducing diabetes (Fig. 1A) . Similarly, application of telmisartan (5 mg/kg) or DHMEQ (5 mg/kg) led to significant (P < 0.01) suppression of retinal angiotensin II and AT1-R protein levels. |
Discussion |
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B pathway with diabetes-induced retinal inflammation. First, the expression and production of the RAS-related molecules were upregulated in the retina when experimental diabetes was induced (Fig. 1) . Second, diabetes-induced leukocyte adhesion to the retinal vasculature was shown to be suppressed by blocking AT1-R, but not AT2-R, signaling (Fig. 2) . Third, the molecular mechanisms in the ARB-induced suppression of retinal leukocyte adhesion included the inhibitory effects on diabetes-induced expression of the inflammatory molecules VEGF and ICAM-1 (Fig. 3) . The inhibition of NF-B nuclear translocation exhibited equivalent effects on these diabetes-induced retinal inflammatory parameters compared with AT1-R blockade (Fig. 4) . An important finding was that in vitro ARB application led to significant suppression of glucose-induced expression of ICAM-1 and MCP-1 via inhibiting NF-B nuclear translocation (Fig. 5) . In addition to its suppressive effect on these inflammatory parameters, the in vivo ARB or DHMEQ treatment attenuated diabetes-induced retinal expression of angiotensin II and AT1-R per se (Fig. 6) .
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Our blocking experiments revealed that diabetes-induced leukocyte adhesion to the retinal vasculature depended on angiotensin II signaling via AT1-R, but not AT2-R (Fig. 2) . This is compatible with our recent reports showing the suppressive effect of the ARB on leukocyte adhesion in hypoxia-induced retinal neovascularization17 and endotoxin-induced retinal inflammation.18 Leukocyte adhesion to the retinal vasculature is a critical antecedent event to vascular hyperpermeability,3 obliteration,4 and proliferation,2 all of which represent the pathogenesis of diabetic retinopathy. The present data indicated AT1-R blockade as a possible therapeutic strategy for preventing leukocyte adhesion and subsequent microvascular complications in diabetic retinopathy. Similarly, acridine orange leukocyte fluorography, capable of evaluating extravasated leukocytes in the retinal tissue,39 revealed the suppressive effect of AT1-R blockade on leukocyte transmigration in the diabetic retina,40 supporting our current data on leukocyte adhesion to the vessel walls (Fig. 2) .
The present in vivo analyses demonstrated the involvement of VEGF and ICAM-1 as molecules responsible for the ARB-induced suppression of leukocyte adhesion in the diabetic retina (Fig. 3) . ICAM-1, constitutively expressed on vascular endothelial cells at a low level, is swiftly upregulated during inflammation, resulting in enhancement of leukocyte–endothelial cell interaction.41 Previous studies of donor eyes from diabetic subjects12 and experimentally induced diabetes5 7 demonstrated that retinal ICAM-1 expression was elevated together with leukocyte adhesion and infiltration. Antibody-based blockade or genetic ablation of ICAM-1 led to significant suppression of vascular hyperpermeability in early diabetes5 or capillary loss in established diabetes.6 VEGF, a potent angiogenic and proinflammatory factor, plays a central role in the pathogenesis of diabetic retinopathy. In patients with diabetic retinopathy, VEGF levels in the intraocular fluid were increased not only during the proliferative stage,42 43 but also during the nonproliferative stage.44 VEGF is also known as the upstream stimulant for ICAM-1 expression in diabetes.3 7 Reasonably, anti-VEGF agents have been applied to eyes with diabetic macular edema in recent clinical trials.45 Angiotensin II levels are elevated and correlated with VEGF levels in the vitreous fluid of patients with diabetic macular edema.37 It has been shown to induce ICAM-146 and VEGF47 via AT1-R in previous in vivo and in vitro studies, supporting the present data on the ARB-induced suppression of these inflammation-related molecules in the diabetic retina (Fig. 3) . In contrast to AT1-R blockade, AT2-R blockade in our present study did not alter retinal expression of VEGF and ICAM-1. In the rodent model of oxygen-induced retinopathy, AT2-R blockade with PD123319 led to significant suppression of VEGF.31 This divergence may be attributable to the difference in stimuli for VEGF induction (i.e., hyperglycemia versus hypoxia/ischemia). Long-term administration of PD123319 with the duration of 4 weeks (1 week in the present study) led to significant suppression of VEGF in the diabetic retina.26 AT2-R is suggested to play a more chronic role in the pathogenesis of diabetic retinopathy than does the AT1-R/NF-B pathway, which causes acute retinal inflammation.18 48 So far, no data have been presented concerning AT2-R's blocking effect on retinal ICAM-1. The present finding that AT2-R blockade did not affect retinal ICAM-1 is compatible with its negligible effect on retinal leukocyte adhesion (Fig. 2) .
Since NF-B is suggested to induce the expression of various inflammatory molecules as a downstream pathway via AT1-R,21 we investigated the proinflammatory role of NF-B in the diabetic retina. Of note, inhibition of NF-B nuclear translocation led to significant suppression of cellular and molecular inflammation-related parameters, including the number of adherent leukocytes and the expression of VEGF and ICAM-1 in the diabetic retina (Fig. 4) , to the similar levels observed in the AT1-R-blocking data (Fig. 3) . NF-B is typically a heterodimer that consists of the p65 and p50 proteins. In the cytoplasm, NF-B is usually inactive without stimulation because of its binding of I-B, which prevents nuclear translocation of NF-B. After I-B phosphorylation and degradation due to various stimuli, NF-B, capable of entering the nucleus and binding the B sequence, promotes the transcription of target genes including VEGF, ICAM-1, and MCP-1.20 Unlike other NF-B inhibitors that block I-B phosphorylation, DHMEQ used in the present study is unique in terms of acting at the level of the nuclear translocation of NF-B.27 Recent studies have established the inhibitory effects of DHMEQ on in vitro cytokine expression49 and in vivo tumor growth and angiogenesis.50 The present data on the role of NF-B in diabetes-induced retinal inflammation (Fig. 4) are compatible with previous studies showing that NF-B inhibition with pyrrolidine dithiocarbamate led to significant suppression of ischemia-induced retinal neovascularization51 and endotoxin-induced ocular inflammation,48 both of which recently have been shown to be mediated by AT1-R.17 18
Hyperglycemia is a primary factor that causes diabetic microangiopathy by enhancing leukocyte–endothelial cell interaction.52 53 To confirm the molecular mechanisms involving the ARB-induced anti-inflammatory effects on the diabetic retina, the in vitro culture system with microvascular endothelial cells stimulated with high glucose was used. AT1-R blockade led to significant suppression of glucose-induced NF-B nuclear translocation to the similar level of the NF-B inhibitor DHMEQ application (Figs. 5A 5B) . NF-B activation was shown to be involved in glucose-induced upregulation of ICAM-153 and MCP-154 in endothelial cells. Our in vitro data showed that the ARB-induced suppressive effects on glucose-induced upregulation of ICAM-1 and MCP-1 expression (Figs. 5C 5D) were mediated by the inhibition of the NF-B pathway. MCP-1 is a member of the C-C chemokine family known as a potent chemoattractant for monocytes and macrophages. Recent clinical data demonstrated that MCP-1 was increased in the vitreous fluid11 and the fibrovascular tissue55 in eyes with proliferative diabetic retinopathy, suggesting that MCP-1 is implicated in the pathogenesis of diabetic retinopathy. In the present in vitro experiments (Fig. 5) , we used vascular endothelial cells derived from the brain. Although the diabetic brain and the retina show several inflammatory changes including the level of ICAM-1 expression56 and leukocyte adhesion,57 the diabetic brain does not develop severe vascular abnormalities as seen in diabetic retinopathy, suggesting a limited interpretation of our in vitro data in brain endothelial cells used to explain the pathogenesis of diabetic retinopathy.
In addition to its suppressive effect on the inflammatory parameters, ARB or DHMEQ treatment attenuated diabetes-induced enhancement of retinal angiotensin II and AT1-R production per se (Fig. 6) . During the tissue RAS activation, angiotensin II was reported to stimulate further production of angiotensinogen via the AT1-R/NF-B pathway, showing a positive feedback loop or vicious cycle.58 AT-1R expression has also been shown to be regulated by NF-B.59 The present data on the suppressive effect of ARB or DHMEQ treatment on the pathogenic ligand-receptor system per se indicates the existence of the RAS/NF-B-mediated vicious cycle in the diabetic retina. This is compatible with our recent report showing the suppression of AT1-R by blocking AT1-R in endotoxin-induced retinal inflammation.29
Collectively, the present in vivo and in vitro findings suggest the AT1-R/NF-B pathway as a therapeutic target to prevent the development of diabetic retinopathy. The use of two different ARBs throughout the current experiments confirmed that the ARB-induced suppression of diabetes-induced retinal inflammation is a class effect. In clinical practice, ARBs are safely and widely used in patients with hypertension. A randomized controlled trial indicated that tight blood pressure control with a ß-blocker or an ACE inhibitor prevents the progression of diabetic retinopathy and deterioration in vision.60 Reasonably, AT1-R blockade is suggested not only to suppress diabetes-induced retinal inflammation but also to improve the systemic background contributing to diabetic retinopathy. The present study is likely to provide a molecular basis for the results in a clinical study that showed that the RAS blockade with an ACE inhibitor results in significant suppression of the progression of diabetic retinopathy.23 It is notable, however, that there are indeed a large number of normotensive patients with diabetic retinopathy who have the potential risk of hypotension caused by antihypertensive agents. NF-B, a downstream mediator of AT1-R without affecting systemic blood pressure (Table 2) , is therefore considered to be an alternative and novel therapeutic target to control several inflammatory molecules related to diabetic retinopathy.
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Supported by Grant-in-aid 17791255 for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (NN).
Submitted for publication December 13, 2006; revised March 15 and May 16, 2007; accepted July 2, 2007.
Disclosure: N. Nagai, Boehringer Ingelheim, Novartis Pharma (F); K. Izumi-Nagai, None; Y. Oike, None; T. Koto, None; S. Satofuka, None; Y. Ozawa, None; K. Yamashiro, None; M. Inoue, None; K. Tsubota, None; K. Umezawa, None; S. Ishida, Boehringer Ingelheim (F), Novartis Pharma (F)
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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B inhibitor. Tetrahedron. 2004;60:7061–7066.[CrossRef][Web of Science]This article has been cited by other articles:
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P < 0.05).
