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Published as doi: 10.1096/fj.07-9377com.
 (The FASEB Journal. 2008;22:1094-1103.)
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(The FASEB Journal. 2008;22:1094-1103.)
© 2008 FASEB

Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis

Kousuke Noda*, Shinsuke Miyahara*, Toru Nakazawa*, Lama Almulki*, Shintaro Nakao*, Toshio Hisatomi*, Haicheng She*, Kennard L. Thomas*, Rebecca C. Garland*, Joan W. Miller*, Evangelos S. Gragoudas*, Yosuke Kawai{dagger}, Yukihiko Mashima{dagger} and Ali Hafezi-Moghadam*,1

* Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston, Massachusetts, USA; and

{dagger} R-Tech Ueno, Ltd., Tokyo, Japan

1Correspondence: Angiogenesis Laboratory, 325 Cambridge St., 3rd Floor, Boston, MA 02114, USA. E-mail: ahm{at}meei.harvard.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammatory leukocyte accumulation is a common feature of major ocular diseases, such as uveitis, diabetic retinopathy, and age-related macular degeneration. Vascular adhesion protein-1 (VAP-1), a cell surface and soluble molecule that possesses semicarbazide-sensitive amine oxidase (SSAO) activity, is involved in leukocyte recruitment. However, the expression of VAP-1 in the eye and its contribution to ocular inflammation are unknown. Here, we investigated the role of VAP-1 in an established model of ocular inflammation, the endotoxin-induced uveitis (EIU), using a novel and specific inhibitor. Our inhibitor has a half-maximal inhibitory concentration (IC50) of 0.007 µM against human and 0.008 µM against rat SSAO, while its IC50 against the functionally related monoamine oxidase (MAO) -A and MAO-B is >10 µM. In the retina, VAP-1 was exclusively expressed in the vasculature, and its expression level was elevated during EIU. VAP-1 inhibition in EIU animals significantly suppressed leukocyte recruitment to the anterior chamber, vitreous, and retina, as well as retinal endothelial P-selectin expression. Our data suggest an important role for VAP-1 in the recruitment of leukocytes to the immune-privileged ocular tissues during acute inflammation. VAP-1 inhibition may become a novel strategy in the treatment of ocular inflammatory diseases.—Noda, K., Miyahara, S., Nakazawa, T., Almulki, L., Nakao, S., Hisatomi, T., She, H., Thomas, K. L., Garland, R. C., Miller, J. W., Gragoudas, E. S., Kawai, Y., Mashima, Y., Hafezi-Moghadam, A. Inhibition of vascular adhesion protein-1 suppresses endotoxin-induced uveitis.


Key Words: semicarbazide-sensitive amine oxidase • ocular inflammation • leukocyte recruitment


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
VASCULAR ADHESION PROTEIN-1 (VAP-1), a 170 kDa homodimeric sialylated glycoprotein, is an endothelial adhesion molecule involved in the leukocyte recruitment cascade (1) . Although VAP-1 was originally discovered in inflamed synovial vessels (1) , it is also expressed on the endothelium of tissues such as skin, brain, lung, liver, and heart under normal and inflamed conditions (2 3 4 5) . In humans, increased levels of both soluble and membrane-associated VAP-1 have been reported in a number of inflammatory diseases, including rheumatoid arthritis, inflammatory bowel disease, diabetes, and atherosclerosis (6) .

Interestingly, VAP-1 regulates leukocyte recruitment under physiological and pathological conditions both as an adhesion molecule and as an enzyme (1 , 7) . VAP-1 has large homology with semicarbazide-sensitive amine oxidases (SSAO), enzymes that catalyze the deamination of primary amines such as methylamine and aminoaceton (8 , 9) . SSAO is a heart-shaped dimer having a unique topaquinone cofactor (a posttranslational modification of an intrinsic tyrosine) that is necessary for the catalytic reaction at the active site (10 , 11) . The active site of SSAO generates toxic formaldehyde and methylglyoxal, hydrogen peroxide and ammonia (9) , reactive chemicals, and major reactive oxygen species (6) . Furthermore, VAP-1 cross-linking causes NF-{kappa}B activation, leading to up-regulation of proinflammatory adhesion molecules and chemokines (12) . Previously, SSAO activity was detected in retinal tissues (13 , 14) . However, its physiological role has not been elucidated. We hypothesized that VAP-1 facilitates the acute inflammatory response in retinal tissues through both its function as an adhesion molecule during leukocyte transendothelial migration and its enzymatic activity.

To study the role of VAP-1 in ocular tissues, we used an established model of acute ocular inflammation, the endotoxin-induced uveitis (EIU; ref. 15 ). EIU is characterized by an acute inflammatory response in the eye, comprised of leukocyte infiltration and protein leakage into the anterior segment, vitreous cavity, and retina (15 16 17 18 19) . As part of this inflammatory response, endothelial adhesion molecules are up-regulated. For example, endothelial P-selectin, which mediates the first step of the leukocyte recruitment, the tethering and rolling, is up-regulated in retinal vessels of EIU animals with a biphasic pattern (18 , 20) . Furthermore, intercellular adhesion molecule-1 (ICAM-1), which mediates the subsequent step of firm leukocyte adhesion to the vascular endothelium, is increased in the retina of EIU animals (21 , 22) . Functional inhibition of P-selectin (19) or ICAM-1 (23) prevents the infiltration of leukocytes into the inflamed ocular tissues during EIU and thus attenuates the inflammatory response at the early stages of rolling and firm adhesion. However, little is known about the molecules that mediate the later stages of recruitment, the transendothelial migration of leukocytes during ocular inflammation. Since VAP-1 is known to facilitate the transendothelial migration of neutrophils and lymphocytes in nonocular tissues (1 , 7) and these cells comprise a majority of the leukocytes found in the affected tissues of EIU animals (15 , 24 , 25) , we hypothesized that VAP-1 may play an important role in the pathogenesis of EIU.

Here, we investigate the role of VAP-1 in the ocular tissues of normal and EIU animals. Furthermore, we elucidate the effect of a novel and specific VAP-1 inhibitor on a variety of inflammatory parameters during acute ocular inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of VAP-1/SSAO expressing transfectants
Molecular cloning of the cDNA encoding human and rat VAP-1/SSAO was carried out by polymerase chain reaction (PCR) with a lung cDNA library (lung QUICK-Clone cDNA, BD Biosciences, Franklin Lakes, NJ, USA), using a modification of the method described by Smith et al. (8) . The amplified cDNA fragment was sequenced using a genetic analyzer (ABI3730, Applied Biosystems, Foster City, CA, USA) and inserted into the expression vector pcDNA3.1(–) (Invitrogen, Carlsbad, CA, USA). The expression vector (42 µg) was used to transfect COS-7 cells using FuGENE 6 (Roche Diagnostics, Basel, Switzerland). Cells were assayed 72 h after transient transfection. The nucleotide sequences of the PCR primers were 5'-CGT TCT AGA ATG AAC CAG AAG ACA ATC CTC-3' (forward) and 5'-CAG CTC GAG CTA GTT GTG AGA GAA GCC CC-3' (reverse) for human VAP-1/SSAO and 5'-CAA TCT AGA ATG CCC AGA AGA CCA CCC-3' (forward) and 5'-TGG CTC GAG TTA TTT ATA AGT AAA GCC CCC-3' (reverse) for rat VAP-1/SSAO.

Amine oxidase assays
COS-7 cells expressing either human or rat VAP-1/SSAO were detached with trypsin-EDTA, washed in culture medium, and resuspended in lysis buffer. The cell extracts were clarified by centrifugation, and the expression of VAP-1/SSAO was examined by immunoblotting with monoclonal antibodies (mAb) ({alpha}-human VAP-1: TK8–14, Serotec, Raleigh, NC, USA; {alpha}-rat VAP-1: Clone 54, BD Biosciences). Total protein concentrations were measured using Quick Start Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA, USA). The cell extracts were used directly in enzyme assays. Monoamine oxidase (MAO) -A and -B were purchased from Sigma (St. Louis, MO, USA).

The half-maximal inhibitory concentration (IC50) of the novel VAP-1 inhibitor, U-V002, a small molecule that selectively reacts with topaquinone, was measured by determining the VAP-1/SSAO activity, using the previously described radioenzymatic method (8 , 14) . Briefly, the extracts containing the VAP-1 protein were preincubated with various concentrations of U-V002 at room temperature for 20 min (8) and subsequently incubated in the presence of [14C]benzylamine (1x10–5 M, 3.7 kBq; GE Healthcare, Little Chalfont, Buckinghamshire, UK) in a final volume of 200 µl of 50 mM potassium phosphate buffer at 37°C for 2 h. The enzyme reactions were terminated with citric acid (200 µl, 2 M). The oxidized products were extracted into 1 ml of toluene/ethyl acetate (1:1, v/v) of which 750 µl was then transferred to a counting vial containing 3 ml Ultima Gold (Perkin Elmer, Wellesley, MA, USA). Radioactivity was assessed using a liquid scintillation counter (Tricarb 2900TR, Perkin Elmer). The VAP-1/SSAO activity was compared to that in the absence of U-V002, and then IC50 values were calculated. The IC50 of U-V002 for MAO-A and MAO-B was measured as described previously (26) .

Endotoxin-induced uveitis
Uveitis was induced in male Lewis rats (8- to 10-wk-old), weighing ~200 g (Charles River Laboratories, Inc.; Wilmington, MA, USA), by injection of 100 µg lipopolysaccharide (LPS; 1 mg/ml) into one hind footpad (15 , 16) . Control animals received footpad injections of the same volume of vehicle (100 µl of saline). All animal experiments were approved by the Animal Care Committee of the Massachusetts Eye and Ear Infirmary.

Treatment
To inhibit VAP-1, we used a specific VAP-1 inhibitor, U-V002, developed and provided by R-Tech Ueno (Tokyo, Japan). U-V002 (0.3 mg/kg) was systemically administered to rats through an intravenous injection, immediately after the LPS injection. As a control, animals received an injection of the vehicle solution (R-Tech Ueno).

Reverse transcription (RT) polymerase chain reaction and real-time PCR
The expression levels of VAP-1 and other endothelial adhesion molecules in retinal tissues of control and EIU rats were examined by RT-PCR and real-time PCR. In brief, the retinal tissues were obtained from control and EIU rat eyes at 24 h after saline or LPS injection and homogenized in extraction reagent (TRIzol Reagent; Invitrogen, Carlsbad, CA, USA). Total RNA was prepared according to the manufacturer’s protocol. Equal amounts of total RNA extracted from samples were reverse-transcribed with a First-Strand cDNA synthesis kit (GE Healthcare) at 37°C for 1 h in a 15 µl reaction volume. Subsequently, PCR was performed using Platinum PCR SuperMix (Invitrogen) with a thermal controller (GeneAmp PCR System 9700; Applied Biosystems). The thermal cycle was 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C, followed by 5 min at 72°C. The reaction was performed for 35 cycles for amplification of VAP-1 and P-selectin, 25 cycles for GAPDH, and 30 cycles for ICAM-1 and vascular cell adhesion molecule-1 (VCAM-1), with previously designed primers (20 , 27 28 29) . PCR products were analyzed by electrophoresis in 1.5% agarose gel and stained with 0.2 µg/ml ethidium bromide.

For quantitative analysis of VAP-1 expression, a real-time PCR assay was performed (TaqMan, with the Prism 7700 Sequence Detection System; Applied Biosystems), according to the manufacturer’s protocol. Furthermore, to examine whether VAP-1 inhibition alters the expression level of endothelial adhesion molecules, P-selectin, ICAM-1, VCAM-1, and VAP-1 expression in EIU retinal tissues were analyzed by real-time PCR, 24 h after the inhibitor administration. Primers and TaqMan probes for rat P-selectin, ICAM-1, VCAM-1, VAP-1 (TaqMan Gene Expression Assays), and GAPDH (Pre-Developed TaqMan Assay Reagents) were purchased from Applied Biosystems. The cycling conditions were 50°C for 2 min, 95°C for 10 min, and 40 cycles at 95°C for 15 s and 60°C for 1 min. The quantity of mRNA expression was calculated by normalizing the threshold cycle (CT) of VAP-1 to the CT of the GAPDH in the same sample, according to the comparative {Delta}{Delta}CT method (30) .

Immunofluorescence microscopy
Expression and localization of VAP-1 protein in the ocular tissues of control and EIU rats were examined by immunofluorescence microscopy. Twenty-four hours after LPS injection, animals were deeply anesthetized with an intramuscular injection of xylazine (10 mg/kg) and ketamine (100 mg/kg). Subsequently, the chest cavity was carefully opened, and a 14-gauge perfusion cannula was introduced into the aorta. A small cut in the right atrium was made to allow drainage. Subsequently, the animals were perfused with PBS [500 ml/kg body weight (BW)], and eyes were enucleated and fixed with 10% neutral buffered formalin overnight. Paraffin-embedded sections were prepared and incubated with blocking solution (Invitrogen). Subsequently, the sections were incubated overnight at 4°C with mouse mAb against rat VAP-1 (1:200; BD Biosciences) or a nonbinding isotype-matched control (R&D systems, Minneapolis, MN, USA). Thereafter, the sections were incubated for 30 min at room temperature with secondary Ab (Alexa Fluor 546, Molecular Probes, Eugene, OR, USA). Sections were mounted with Vectashield mounting media with 4',6-diamino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA), and photomicrographs were taken with an x20 objective on a fluorescent microscope (DM RXA; Leica, Solms, Germany).

SSAO activity measurements
The control and EIU animals were perfused with PBS (500 ml/kg BW) under deep anesthesia, and eyes were enucleated at 24 h after LPS or saline injection. Subsequently, retinas were isolated and homogenized (1:10, w/v) in 50 mM potassium phosphate buffer (pH 7.4) using an Ultra-Turrax (IKA, Staufen, Germany) (10 s at 30,000 rpm). Crude homogenates were centrifuged at 800 g for 10 min at 4°C, and the supernatant fractions were used for the assay. Protein concentration was determined by Quick Start Protein Assay Kit (Bio-Rad Laboratories). The SSAO enzyme preparations were preincubated with pargyline (0.5 mM, Sigma) at room temperature for 20 min to inhibit MAO activity. Subsequently, the enzyme preparation was incubated in the presence of [14C]benzylamine (1x10–5 M, 3.7 kBq) and the SSAO activity was determined radiochemically.

Analysis of inflammatory cells and protein content in the aqueous humor
Aqueous humor was collected by anterior chamber puncture using a 30-gauge needle under a surgical microscope at 4, 10, 24, and 48 h after the LPS treatment. The samples were used for either cell counting or protein concentration measurements. To quantify the number of infiltrated leukocytes, the samples were suspended with an equal volume of 0.4% trypan blue stain solution, and the cells per field (an equivalent of 0.1 µl) were manually counted using a hemocytometer under a light microscope (Leica) in a masked fashion. The protein concentration in the aqueous humor was determined using a protein assay kit (Bio-Rad Laboratories) and dilutions of bovine serum albumin as standards.

Analysis of leukocyte infiltration into the vitreous cavity
The number of infiltrated leukocytes into the vitreous cavity was analyzed as described previously (16) . Briefly, 24 h after the injection of the inhibitor or vehicle solution to EIU animals, eyes were enucleated from the animals under deep anesthesia and fixed in 4% paraformaldehyde. After fixation, tissues were processed and embedded in paraffin using standard techniques. Three five micrometer sections were prepared at a distance of 100 µm to each other with the middle section passing through the optic nerve. All sections were stained with hematoxylin and eosin, and the number of infiltrating cells in the vitreous cavity was counted.

Leukocyte migration in the retina
Leukocyte migration in the retinal tissues was investigated by the two distinct techniques of in vivo acridine orange (AO) staining (17) and immunofluorescent microscopy (31) .

In vivo AO staining
The rate of leukocyte transmigration at different time points was investigated by in vivo AO staining (17) . AO (Sigma), which fluorescently stains intravascular leukocytes, endothelial cells, and migrated leukocytes, was injected into the rats via tail vein at 4, 10, 24, and 48 h after VAP-1 inhibitor or vehicle treatment. Although the concentration of AO in the intravascular leukocytes and the endothelial cells significantly diminishes by 30 min after AO injection due to the washout effect (17) , the transmigrated leukocytes in the retinal parenchyma retain the staining and can thus be quantified (17) . To investigate the rate of transmigration, animals were euthanized with an overdose of anesthesia 30 min after the AO injection. Subsequently, eyes were enucleated and retinal flatmounts were prepared using a mounting medium (Vector Laboratories). The number of fluorescent dots in the retina within four separate circles of 800 µm diameter adjacent to the optic disc were counted using fluorescence microscopy (DM RXA; Leica) and averaged for each retina (17) .

Immunofluorescence
In addition to the evaluation of the rate of retinal leukocyte extravasation using AO, the cumulative number of transmigrated leukocytes was visualized by immunofluorescence microscopy. EIU animals were treated with the inhibitor or vehicle, and the globes were enucleated after euthanasia at 24 and 72 h after LPS injection. Frozen sections of 10 µm thickness from the posterior segment were prepared and preblocked (PBS containing 10% goat serum, 0.5% gelatin, 3% BSA, and 0.2% Tween 20). The sections were incubated with mouse {alpha}-CD45 mAb (1:100; BD Pharmingen, San Diego, CA, USA) and {alpha}-GFAP Ab (1:100; Dako North America, Inc., Carpinteria, CA) and subsequently incubated with the secondary Ab (Invitrogen). Sections were mounted with Vectashield mounting media with DAPI (Vector Laboratories). The photographs of retinal sections were taken with an x20 objective on a fluorescent microscope (DMRXA, Leica), and the number of CD45+ cells was counted in the section images. Six sections were prepared from each eye, and the averages from the leukocyte numbers were used.

Quantification of firm leukocyte adhesion
Animals were perfused with 100 ml of PBS/kg BW to remove intravascular content, including nonadherent leukocytes. Perfusion with concanavalin A (ConA, 40 µg/ml in PBS pH 7.4, 5 mg/kg BW) was then performed to label adherent leukocytes and vascular endothelial cells, followed by removal of residual unbound lectin with PBS perfusion. The retinas were carefully removed and flatmounted in a mounting medium (Vector Laboratories). Each retina was imaged with a fluorescence microscope (DM RXA; Leica), and the total number of adherent leukocytes per retina was counted.

Measurement of retinal vessel diameters
Animals were anesthetized and the pupils were dilated with 0.5% tropicamide and 2.5% phenylephrine hydrochloride. Monochromatic retinal images were recorded by scanning laser ophthalmoscope (SLO; HRA2; Heidelberg Engineering, Dossenheim, Germany) coupled with a computer-assisted image analysis system. Subsequently, the diameters of all major retinal vessels were measured as described previously (20) .

Statistical analysis
All results are expressed as mean ± SE with n numbers as indicated. Student’s t test was used for statistical comparison between the groups. To compare three or more conditions, statistical analysis was performed by post hoc comparisons with the Fisher’s protected least significant difference procedure. Differences between the means were considered statistically significant when the probability values were <0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Specificity of the VAP-1 inhibitor
To examine the specificity of the novel VAP-1 inhibitor U-V002, we quantified SSAO enzymatic activity in a radiochemical assay. In this assay, [14C]benzylamine is used as substrate, and the ratio IC50 values for SSAO and the functionally related MAO inhibition were obtained as an indication of the selectivity of our inhibitor. U-V002 showed an IC50 of 0.007 µM against human and 0.008 µM against rat SSAO, while its IC50 against human MAO-A and MAO-B were >10 µM. This indicates a specific inhibition of both human and rat SSAO activity of VAP-1 but not MAO-A and MAO-B.

VAP-1 expression in the retina during EIU
To investigate the expression of VAP-1 in the retinal tissue of normal and EIU animals, we analyzed VAP-1 mRNA levels by PCR. VAP-1 mRNA was constitutively expressed in retinal tissues under normal conditions and up-regulated in the retinas of EIU animals (Fig. 1 A). In line with previous studies (16 , 18 , 20 , 22) , P-selectin, ICAM-1, and VCAM-1 were up-regulated 24 h after LPS injection, confirming the acute inflammatory response in the retinal tissues of these animals (Fig. 1A ). Real-time PCR showed a 1.5-fold higher VAP-1 expression in the retinas of EIU animals 24 h after LPS injection compared to that of normal animals, suggesting that VAP-1 is up-regulated in the retinal tissues during the acute inflammatory response (n=10; P<0.05; Fig. 1B ).


Figure 1
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  		Figure 1. Expression, localization, and enzymatic activity of VAP-1 in retinal tissues. A) RT-PCR amplification of adhesion molecule mRNAs from retinas of EIU rats, 24 h after LPS injection. VAP-1 mRNA was constitutively expressed in retinal tissue under normal conditions and up-regulated 24 h after LPS stimulation. B) Quantitative real-time PCR analysis showing increased retinal VAP-1 expression in EIU animals compared to normal controls. Values are mean ± SE (n=10). *P < 0.05. C) Immunolocalization of VAP-1 protein in the retina. Fluorescent micrograph of retinal tissues of a representative control animal (n=3), showing VAP-1 immunostaining (red) and DAPI counterstained nuclei (blue). Scale bar = 50 µm. VAP-1 was exclusively expressed in the endothelial cells of retinal vessels, showing a dot-like distribution pattern. D) SSAO activity in retinal tissues of control and EIU animals with or without VAP-1 inhibition at 24 h after saline or LPS treatment (n=5–8). The enzymatic activity of VAP-1/SSAO in the retinal extracts was measured by radiochemistry. Values are mean ± SE. *P < 0.05; {dagger}P < 0.01.

To confirm the presence of VAP-1 in retinal tissues on the protein level, we conducted immunofluorescence staining of VAP-1. In the retinal tissues, the signal for VAP-1 was exclusively found in the vascular endothelial cells, showing a dot-like distribution pattern (Fig. 1C ). The immunofluorescence staining supports the real-time PCR data, showing constitutive VAP-1 expression in the retina. However, the intensity of the immunofluorescence signal appeared unchanged in EIU animals compared to that in normal controls (data not shown).

SSAO activity in the retina
To investigate the SSAO enzymatic activity of VAP-1 in the eye, we harvested retinal tissues from normal control and EIU animals. In normal saline-treated animals, there was a constitutive level of SSAO activity (1.01±0.1 pmol/min/mg protein; n=8), which was significantly increased 24 h after LPS-injection (1.98±0.38 pmol/min/mg protein; n=8, P<0.05; Fig. 1D ). In contrast, EIU animals that were treated with the VAP-1 inhibitor showed significantly lower retinal SSAO activity (0.15±0.02 pmol/min/mg protein; n=5; P<0.01) compared to untreated EIU animals or normal controls (P<0.01; Fig. 1D ), indicating the bioavailability of the compound in the eye and its potency for reduction of the enzymatic activity of VAP-1 in vivo.

Role of VAP-1 in the anterior uveitis
To evaluate the role of VAP-1 in the anterior uveitis, we examined leukocyte infiltration and protein leakage into the aqueous humor in EIU animals with and without specific inhibition of VAP-1. In vehicle-treated EIU rats, both the average leukocyte count and protein concentration gradually increased after LPS treatment with a peak at 24 h (Fig. 2 ). In contrast, inhibition of VAP-1 in the EIU animals with the inhibitor significantly reduced the number of infiltrated cells (n=6 at each time point; P<0.05 in post hoc comparisons; Fig. 2A ) and the protein concentration in the aqueous humor (n=6 at each time point; P<0.01, post hoc comparisons; Fig. 2B ). The largest amount of reduction was measured 24 h after LPS injection, when VAP-1 inhibition significantly reduced leukocyte infiltration into the anterior chamber by 58.7% (3097±882 vs. 1280±298 cells/µl; n=6; P<0.05) and protein leakage into the aqueous humor by 46% (32.4±2.9 vs. 17.5±2.2 mg/ml; n=6; P<0.01), compared to the vehicle-treated controls (Fig. 2) .


Figure 2
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  		Figure 2. Role of VAP-1 in inflammation in the aqueous humor and vitreous. Inflammatory leukocyte infiltration (A) and protein leakage (B) into the aqueous humor of EIU animals at 24 h after LPS injection with or without VAP-1 inhibition. The novel VAP-1 inhibitor U-V002 (0.3 mg/kg BW) or vehicle was administered immediately after LPS injection. Aqueous humor was collected at the indicated times. The cell number and protein concentration are mean ± SE (n=6 at each time point). *P < 0.05; {dagger}P < 0.01. C) Representative micrographs of leukocyte infiltration into the vitreous of EIU animals with or without VAP-1 inhibitor treatment. D) The number of infiltrated cells in the vitreous cavity of EIU animals was decreased by VAP-1 inhibition. Values are mean ± SE (n=6 in each group). *P < 0.05.

To study the role of VAP-1 in leukocyte extravasation into the vitreous cavity, we prepared paraffin sections of whole eyes from normal and EIU animals with or without inhibitor treatment and quantified the number of leukocytes in these sections. Representative sections from these animals show extravasated leukocytes around the optic disc (Fig. 2C ). Whereas normal animals show no or very few leukocytes in their vitreous cavity (16) , there was a high number of leukocytes in the vitreous of EIU animals (74±10 cells; n=6). However, when VAP-1 was inhibited in the EIU animals, the number of infiltrating cells into the vitreous cavity was significantly reduced by 45.3% compared with the vehicle-treated group (41±9 cells; n=6; P<0.05; Fig. 2D ).

Role of VAP-1 in retinal leukocyte transmigration
To elucidate the role of VAP-1 during retinal leukocyte infiltration, we quantified the rate and cumulative number of transmigrated leukocytes in the retinal tissues of EIU animals by two different methods, AO and immunofluorescent staining of CD45. AO-stained transmigrated leukocytes emitted a bright fluorescent signal in flat-mounted retinas (Fig. 3 A). The peak of the leukocyte transmigration rate in the retinas of the EIU animals was at 10 h (43±7 cells/mm2; n=6), which was significantly reduced with the inhibition of VAP-1 (26±3 cels/mm2; n=6; P<0.05; Fig. 3B ). Quantification of AO staining showed a biphasic pattern of leukocyte migration in the vehicle-treated group, with peaks at 10 and 48 h after LPS injection (Fig. 3B ). However, in the VAP-1 inhibitor-treated group, the second phase of the retinal leukocyte migration was significantly suppressed, leaving a single peak at 10 h after LPS injection. The retinal leukocyte extravasation in the VAP-1 inhibitor treated rats was significantly reduced by 69.8, 39.5, 45.3, and 59.1% at 4, 10, 24, and 48 h after LPS injection, respectively (P<0.05, post hoc comparisons; Fig. 3B ).


Figure 3
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  		Figure 3. Impact of VAP-1 inhibition on the retinal leukocyte extravasation rate during EIU. A) Representative micrographs of the flatmounted retinas obtained from control and EIU animals (10 h after LPS injection) with or without VAP-1 inhibitor treatment: a) no LPS; b) +LPS, +vehicle; and c) +LPS, +VAP-1 inhibitor. Arrows depict transmigrated leukocytes into the retinal tissues 30 min after AO injection. Scale bar = 100 µm. B) Time course of leukocyte transmigration rate. Each data point represents the average number of leukocytes that have transmigrated into the retinal tissues within 30 min at various time points after LPS injection, with or without VAP-1 inhibitor treatment. Values are mean ± SE (n=6 at each time point). *P < 0.05; {dagger}P < 0.01.

In line with the AO data, the number of the cells positive for CD45, a marker for all peripheral blood leukocytes, was significantly decreased by 94% in the inhibitor-treated group at 24 h after LPS injection (3±1 cells/section; n=6; P<0.01), when compared with vehicle-treated controls (51±15 cells/section; n=6), thus confirming the importance of VAP-1 for leukocyte transmigration to the ocular tissues (Fig. 4 ).


Figure 4
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  		Figure 4. Impact of VAP-1 inhibition on retinal leukocyte accumulation during EIU. A) Representative micrographs of retinal tissue sections obtained from control and EIU animals (24 h after LPS injection) with or without VAP-1 inhibitor treatment: a) no LPS; b) +LPS, +vehicle; and c) +LPS, +VAP-1 inhibitor. The retinal sections were stained for CD45 (green), GFAP (red), and DAPI (blue) to reveal the nuclei. Arrows indicate CD45 positive cells in the retina. Scale bar = 100 µm. B) The number of accumulated leukocytes in the EIU retina with or without VAP-1 inhibitor treatment. Values are mean ± SE (n=6 in each group). *P < 0.05.

Reduction of endothelial adhesion molecule expression and leukostasis with VAP-1 inhibition
To investigate whether VAP-1 function during uveitis may perpetuate the inflammatory outcome, we measured in retinal tissues of EIU animals the mRNA expression levels of the endothelial adhesion molecules, P-selectin, ICAM-1, VCAM-1, and VAP-1 after treatment with the VAP-1 inhibitor or control. In the VAP-1 inhibitor-treated animals, the level of retinal P-selectin expression was significantly decreased by 73% at 24 h after LPS injection, when compared with the vehicle-treated EIU animals (n=6; P<0.05; Fig. 5 A). In comparison, ICAM-1 mRNA expression was down-regulated by 37.2%; however, the difference did not reach statistical significance (P=0.1). The expression levels of VCAM-1 and VAP-1 were unchanged between the inhibitor and vehicle-treated groups (P=0.3 and P=0.4, respectively; Fig. 5A ).


Figure 5
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  		Figure 5. Role of VAP-1 inhibition on adhesion molecule expression and firm leukocyte adhesion in the retinal vessels during EIU. A) Quantitative real-time PCR analysis for the endothelial adhesion molecules, P-selectin, ICAM-1, VCAM-1, and VAP-1 in the retinas of EIU animals, 24 h after LPS injection and vehicle or VAP-1 inhibitor treatment. Bars represent fold changes of the retinal expression levels of the endothelial adhesion molecule mRNAs in inhibitor-treated animals compared with vehicle-treated animals. Values are mean ± SE (n=6 in each group). *P < 0.05; N.S. = not significant. B) Bars represent the average numbers of firmly adhering leukocytes in retinal vessels of EIU animals, treated with VAP-1 inhibitor or control from ConA-stained whole retinal flat-mounts. Error bars represent SE (n=7 in each group), *P < 0.05. C) Representative micrographs of flatmounted retinas from EIU rats at 24 h after treatment with vehicle control (a) or the specific VAP-1 inhibitor (b), and high-magnification images of retinal veins from EIU rats after treatment with vehicle (c) or VAP-1 inhibitor-treated (d). Firmly adhering leukocytes in the retinal vasculature were visualized by perfusion with ConA. Arrows distinguish firmly adhering leukocytes in the inflamed retinal vasculature (a, b). Scale bars = 100 µm (a, b); 50 µm (c, d).

To investigate the role of VAP-1 in the leukocyte recruitment cascade, we next quantified firm leukocyte adhesion in the inflamed retinal vessels of untreated and inhibitor-treated EIU animals using the ConA-staining technique (Fig. 5B, C ). Twenty-four hours after LPS-injection, a large number of leukocytes firmly adhered to the retinal vessels (756±125 cells/retina; n=7; P=0.01) compared to controls (66±11 cells/retina; n=4). However, when the EIU animals were treated with the specific inhibitor of VAP-1, the number of firmly adhering leukocytes was significantly reduced by 39.9% (455±107cells/retina; n=7; P<0.05) in comparison with the vehicle-treated controls (Fig. 5B, C ). These data suggest that VAP-1 inhibition may lead to lower numbers of firm leukocyte adhesion either directly through the suppression of VAP-1 function or indirectly through the diminished expression of the other endothelial adhesion molecules, such as P-selectin and ICAM-1.

Impact of VAP-1 inhibition on retinal vessel diameter during acute inflammation
To investigate the role of VAP-1 during acute inflammation on an organ level, we measured the diameter of the retinal vessels in normal and EIU animals with or without inhibitor treatment. The diameter of the retinal veins of EIU animals, 24 h after LPS injection, was significantly larger than the corresponding retinal veins in normal animals (49.5±1.5 µm, n=6, and 33.5±0.9 µm, n=6, respectively; P<0.01; Fig. 6 ). When animals were treated with the VAP-1 inhibitor, the diameter of the retinal veins was significantly reduced 24 h after LPS injection (39.9±1.8 µm; n=6; P<0.05) and at 48 h after LPS injection (38.3±2.1 vs. 44.1±1.9 µm; n=6; P<0.05), compared to the vehicle-treated controls. VAP-1 inhibition also reduced the diameter of retinal arteries 24 h after LPS injection (27.7±0.53 µm; n=6), compared with vehicle-treated rats (30.5±1.36 µm; n=6), but the difference did not reach statistical significance (P=0.1).


Figure 6
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  		Figure 6. VAP-1 inhibition reverses clinical signs of inflammation in the retina during EIU. A) Representative fundus photographs of control rats (a) and EIU rats at 24 h after treatment with vehicle (b) or VAP-1 inhibitor (c). B) Diameters of major retinal vessels of EIU rats, treated with VAP-1 inhibitor or vehicle. Values are mean ± SE (n=6 at each time point); *P < 0.05; {dagger}P < 0.01.

Our data indicate that VAP-1 inhibition substantially suppresses retinal inflammation during EIU on a molecular, cellular, and organ level.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we describe the expression and localization of VAP-1 in normal ocular tissues, its role in acute ocular inflammation, and the therapeutic effect of a novel and specific VAP-1 inhibitor in experimental uveitis. To our knowledge, this is the first report of VAP-1 involvement in ocular inflammation.

Consistent with previous studies (18 , 20 21 22) , we show induction of P-selectin and ICAM-1 mRNA in the retina 24 h after EIU induction. VAP-1 mRNA is constitutively expressed in the normal retina, and its expression is increased during acute inflammation. In accord with our mRNA data, SSAO activity is elevated 1.5-fold in the retinas of EIU animals compared to controls. Furthermore, our immunofluorescence study confirms the constitutive expression of VAP-1 protein and shows the localization of VAP-1 in the retinal endothelium. Under normal conditions, VAP-1 resides in discrete granules within the cytoplasm of endothelial cells and translocates to the surface during inflammation, i.e., in the high endothelial venules (4) . Whether and to what extent intracellular translocation also occurs in the barrier-privileged retinal endothelial cells during EIU remain to be investigated. Our data suggest that during acute inflammation VAP-1 function in the retina is at least in part regulated through de novo synthesis.

Our VAP-1 inhibitor, U-V002, potently and selectively inhibits VAP-1/SSAO enzymatic activity but not the activity of the VAP-1/SSAO-related enzyme, MAO-B. In addition, the inhibitor suppresses the up-regulated SSAO activity in the retinal tissues of EIU animals, indicating its pharmacological effectiveness in vivo.

During inflammation, leukocytes are recruited in a cascade-like fashion, starting with rolling, followed by firm adhesion and extravasation (32 , 33) . Since VAP-1 is thought to mainly play a role during the extravasation step of the cascade (1 , 7) , it appeared likely that VAP-1 inhibition would preferentially inhibit leukocyte-transendothelial migration into the inflamed ocular tissues. Indeed, VAP-1 inhibition markedly reduces the accumulation of infiltrated leukocytes into the aqueous humor and the vitreous cavity. Furthermore, it reduces protein concentration in the aqueous humor, suggesting that the new inhibitor may prevent disruption of the blood-aqueous barrier during anterior ocular inflammation. Notably, VAP-1 contributes to inflammation not only through its role as an adhesion molecule but also through its function as an enzyme by causing the formation of cytotoxic molecules such as hydrogen peroxide, formaldehyde, and ammonia (9) . These molecules are known to be involved in the pathophysiology of ocular inflammation (34 , 35) , and their inhibition, for instance through antioxidants, recovers the integrity of the blood-aqueous barrier in EIU animals (36) . These findings indicate the potential of VAP-1 as a therapeutic target in the treatment of anterior uveitis.

In addition, our AO experiments demonstrate the importance of VAP-1 for extravasation of leukocytes into the retina, since VAP-1 inhibition with U-V002 caused a marked reduction in the rate of retinal leukocyte infiltration at all measured time points. The biphasic pattern of the retinal leukocyte infiltration rate in our experiments is consistent with previous studies (17 , 37) of leukocyte rolling flux in the retinal vessels. The second phase was significantly diminished by VAP-1 inhibition, suggesting that one systemic injection of the VAP-1 inhibitor sustains its pharmacological effect up to 48 h. Alternatively, it is feasible that the suppression of the first peak through VAP-1 inhibition may attenuate the subsequent inflammatory response in the retinal tissues, thereby suppressing the second peak of leukocyte transmigration.

Furthermore, the direct immunofluorescence staining of leukocytes is in accord with our AO data, showing a substantial inhibition of leukocyte accumulation in the inhibitor-treated EIU animals. These data suggest that VAP-1 inhibition may have beneficial effects during experimental uveitis by preventing leukocyte infiltration into the retinal parenchyma. Consistent with our leukocyte recruitment results, we find that the dilation of retinal veins, a general sign of acute inflammation, is significantly reduced by VAP-1 inhibition.

We find that VAP-1 inhibition down-regulates P-selectin mRNA expression in the retinas of EIU animals. Our finding is in line with the recent study (38) of VAP-1 regulatory function in endothelial selectin expression. The expression and complex interplay of a variety of specialized molecules, including cytokines, chemokines, and adhesion molecules, regulate the inflammatory outcome, for instance, during the recruitment cascade. Tumor necrosis factor (TNF)-{alpha}, a proinflammatory cytokine up-regulated during EIU, induces the expression of P-selectin (39) and ICAM-1 (40 , 41) . In addition, hydrogen peroxide, a metabolite of VAP-1/SSAO enzymatic reaction, augments ICAM-1 expression in endothelial cells (40 , 42) . Hydrogen peroxide and formaldehyde generate hydroxyl free radicals, which oxidize lipoproteins in blood (9) . Oxidized lipoproteins are known to induce P-selectin expression (43) . Therefore, the reduced P-selectin and the trend to reduced ICAM-1 expression, which we find in EIU animals, may be in part due to reduced hydrogen peroxide generation and less oxidative stress, when VAP-1 function is inhibited. This hypothesis is further supported by our finding that VAP-1 inhibition significantly reduces the number of firmly adhering leukocytes in the inflamed retinal vessels. This suggests that VAP-1 inhibition also attenuates leukocyte recruitment indirectly through suppression of the expression of other adhesion molecules, such as P-selectin or perhaps also ICAM-1.

In summary, VAP-1 is crucially involved in leukocyte infiltration into ocular tissues during experimental uveitis. It is likely functionally up-regulated via de novo synthesis during EIU and potentially through translocation to the endothelial surface from intracellular compartments. VAP-1 inhibition with a novel and specific inhibitor potently suppresses inflammation during EIU and down-regulates P-selectin expression. Thus VAP-1 inhibition may even prevent leukocyte recruitment at the early stage of rolling. These results suggest VAP-1 inhibition as a novel and potent therapeutic strategy in the treatment of ocular inflammatory diseases.


   ACKNOWLEDGMENTS
 
This work was supported by U. S. National Institutes of Health grant AI-050775 (to A.H.M.), research funds from RTEC-UENO, Bausch & Lomb Vitreoretinal Fellowships (to K.N., S.M., and T.N.), a Research Fellowship Award from Bausch & Lomb and a Fellowship Award from the Japan Eye Bank Association (to S.N.), and NEI core grant EY14104. We are indebted to the Massachusetts Lions Eye Research Fund Inc. for generous funds provided for laboratory equipment used in this project. We thank Research to Prevent Blindness for unrestricted funds awarded to the Department of Ophthalmology at Harvard Medical School. We thank Norman Michaud and Sreedevi Mallemadugula (Massachusetts Eye and Ear Infirmary) for skillful technical assistance in the preparation of the histological sections. We thank Alexander Schering for help with the preparation of the manuscript. We thank Dr. Susan E. Leeman for a critical reading of the manuscript.

Received for publication July 10, 2007. Accepted for publication October 11, 2007.


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