Vascular amine oxidases are needed for leukocyte extravasation into inflamed joints in vivo.
ABSTRACT Leukocyte traffic from the blood to the joints is crucial in the pathogenesis of arthritis. A bifunctional endothelial cell-surface glycoprotein, AOC3 (amine oxidase, copper-containing 3; also known as vascular adhesion protein 1), has both adhesive and enzymatic properties. We undertook this study to determine the contribution of AOC3 and its oxidase activity to leukocyte trafficking into inflamed joints in vivo.
We used gene-modified animals, molecular modeling, an AOC3 enzyme inhibitor, oxidase assays, and arthritis models (adjuvant-induced arthritis [AIA] in rats and anti-type II collagen antibody-induced arthritis in mice) to dissect the importance of AOC3 in vivo.
The AOC3 inhibitor fitted well with a covalent binding mode into the active site of the AOC3 crystal structure. It selectively blocked the oxidase activity of AOC3 in enzyme assays. Intraperitoneal and oral administration of the AOC3 inhibitor significantly ameliorated rat AIA. In anti-type II collagen antibody-induced arthritis in mice, the AOC3 inhibitor also improved the outcome of the joint inflammation. The acute semicarbazide-sensitive amine oxidase blockade by the inhibitor had even more pronounced effects than genetic deletion of AOC3. Enzymatic analyses showed that the inhibitor also blocked 2 other structurally very closely related AOCs, but not any of more than 100 other enzymes tested.
These are the first data to demonstrate that the enzymatic activity of the atypical endothelial adhesion molecule AOC3, and possibly that of other closely related ecto-oxidases, is crucial for leukocyte exit from the vessels in inflamed joints in vivo.
- SourceAvailable from: virginia.eduNew England Journal of Medicine 11/2000; 343(14):1020-34. · 51.66 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The ability to home to extralymphoid tissues, such as the skin, lungs and gut, is acquired by many effector and memory T cells after initial antigen encounter. This review discusses the role of E-, P- and L-selectin and their glycoprotein ligands in this process. During activation, some T cells differentiate to bind E-selectin through induced expression of fucosyltransferase-VII and to bind P- and L-selectin through additional expression of core 2 glucosaminyltransferase-I. This inducible expression of selectin ligands, together with the regulation of L-selectin expression, has a role in the recruitment of activated T cells to extralymphoid tissues and sites of inflammation.Nature reviews. Immunology 06/2004; 4(5):325-35. · 33.13 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Understanding the molecular basis of lymphocyte homing to lymphoid organs was originally a problem of concern only to immunologists. With the discovery of L-selectin and its ligands, interested scientists have expanded to include glycobiologists, immunopathologists, cancer biologists, and developmental biologists. Going beyond its first discovered role in homing to lymph nodes, the L-selectin system is implicated in such diverse processes as inflammatory leukocyte trafficking in both acute and chronic settings, hematogenous metastasis of carcinoma cells, effector mechanisms for inflammatory demyelination of axons, and implantation of the early mammalian embryo. This review focuses on the ligands for L-selectin that are found on vascular endothelium, leukocytes, carcinoma cells, and at various extravascular sites. The discovery of selectins and their ligands has validated the long-predicted hypothesis that carbohydrate-directed cell adhesion is relevant in eukaryotic systems. Emphasis will be given to the carbohydrate and sulfation modifications of the ligands, which enable recognition by L-selectin. The rapid "homing" of labeled cells into the lymph nodes presumably had its basis in the special affinity of small lymphocytes for the endothelium of the postcapillary venules.Annual Review of Immunology 02/2004; 22:129-56. · 36.56 Impact Factor
ARTHRITIS & RHEUMATISM
Vol. 54, No. 9, September 2006, pp 2852–2862
© 2006, American College of Rheumatology
Vascular Amine Oxidases Are Needed for
Leukocyte Extravasation Into Inflamed Joints In Vivo
Fumiko Marttila-Ichihara,1David J. Smith,2Craig Stolen,1Gennady G. Yegutkin,1Kati Elima,1
Nathalie Mercier,1Riku Kiviranta,3Marjo Pihlavisto,2Sakari Alaranta,2Ulla Pentika ¨inen,4
Olli Pentika ¨inen,5Ferenc Fu ¨lo ¨p,6Sirpa Jalkanen,1and Marko Salmi1
Objective. Leukocyte traffic from the blood to the
joints is crucial in the pathogenesis of arthritis. A
bifunctional endothelial cell–surface glycoprotein,
AOC3 (amine oxidase, copper-containing 3; also known
as vascular adhesion protein 1), has both adhesive and
enzymatic properties. We undertook this study to deter-
mine the contribution of AOC3 and its oxidase activity
to leukocyte trafficking into inflamed joints in vivo.
Methods. We used gene-modified animals, molec-
ular modeling, an AOC3 enzyme inhibitor, oxidase
assays, and arthritis models (adjuvant-induced arthritis
[AIA] in rats and anti–type II collagen antibody–
induced arthritis in mice) to dissect the importance of
AOC3 in vivo.
Results. The AOC3 inhibitor fitted well with a
covalent binding mode into the active site of the AOC3
crystal structure. It selectively blocked the oxidase
activity of AOC3 in enzyme assays. Intraperitoneal and
oral administration of the AOC3 inhibitor significantly
ameliorated rat AIA. In anti–type II collagen antibody–
induced arthritis in mice, the AOC3 inhibitor also
improved the outcome of the joint inflammation. The
acute semicarbazide-sensitive amine oxidase blockade
by the inhibitor had even more pronounced effects than
genetic deletion of AOC3. Enzymatic analyses showed
that the inhibitor also blocked 2 other structurally very
closely related AOCs, but not any of more than 100
other enzymes tested.
Conclusion. These are the first data to demon-
strate that the enzymatic activity of the atypical endo-
thelial adhesion molecule AOC3, and possibly that of
other closely related ecto-oxidases, is crucial for leuko-
cyte exit from the vessels in inflamed joints in vivo.
Leukocyte exit from the blood into tissues is
orchestrated by multiple adhesive interactions between
blood-borne leukocytes and the vascular lining. Many
steps of this cascade are governed by adhesion and
activation molecules (1). Selectins and their oligo-
saccharide-based counterreceptors can account for ini-
tial tethering and rolling (2,3). Thereafter, binding of
chemokines to their 7-transmembrane receptors on leu-
kocytes can induce signals, which result in the activation
of integrins that allows shear-resistant firm adhesion of
leukocytes to the vessel wall (4,5). Finally, integrins,
immunoglobulin superfamily members, and other mole-
cules mediate the transmigration of the leukocyte
through the vessel wall into the tissue (6–8).
AOC3 (amine oxidase, copper-containing 3; also
known as vascular adhesion protein 1 [VAP-1]) (EC
188.8.131.52) is a new type of adhesion molecule with 2
interrelated functions (9,10). It belongs to the cell
surface–expressed, semicarbazide-sensitive amine oxi-
dase (SSAO) family (11). These enzymes catalyze oxi-
dative deamination of primary amines in a reaction
resulting in the production of the corresponding alde-
hyde, H2O2, and ammonium (10,12,13). On endothelial
cells, AOC3 can serve as a traditional adhesion molecule
Supported by the Finnish Academy and the Sigrid Juselius
Foundation. Dr. Stolen’s work was supported by the NIH (grant
1Fumiko Marttila-Ichihara, MD, PhD, Craig Stolen, PhD,
Gennady G. Yegutkin, PhD, Kati Elima, MD, PhD, Nathalie Mercier,
PhD, Sirpa Jalkanen, MD, PhD, Marko Salmi, MD, PhD: University
of Turku, and National Public Health Institute, Turku, Finland;2David
J. Smith, PhD, Marjo Pihlavisto, PhD, Sakari Alaranta, PhD: Biotie
Therapies Corporation, Turku, Finland;3Riku Kiviranta, MD, PhD:
University of Turku, Turku, Finland;4Ulla Pentika ¨inen, PhD: Univer-
sity of Turku, and Åbo Akademi University, Turku, Finland;
Pentika ¨inen, PhD: Åbo Akademi University, Turku, Finland;6Ferenc
Fu ¨lo ¨p, PhD: University of Szeged, Szeged, Hungary.
Drs. Stolen, Jalkanen, and Salmi own stock in Biotie Thera-
pies Corporation. Drs. Jalkanen and Salmi formerly held a patent on
vascular adhesion protein 1. Drs. Smith, Pihlavisto, and Alaranta own
stock and/or hold stock options in Biotie Therapies Corporation.
Address correspondence and reprint requests to Marko
Salmi, MD, MediCity Research Laboratory, Tykisto ¨katu 6A, 20520
Turku, Finland. E-mail: email@example.com.
Submitted for publication October 6, 2005; accepted in re-
vised form May 31, 2006.
whose function is blocked by monoclonal antibodies
(mAb) (14–19). On the other hand, the enzymatic
activity of AOC3, which is not inhibited by the anti-
AOC3 mAb, also appears to be critical for its adhesive
functions in flow-based assays in vitro (16,17,20). En-
zyme inhibitors targeting AOC3 may therefore offer
new ways of preventing harmful inflammation in vivo.
We have recently shown that AOC3-deficient
mice display diminished inflammatory responses in vivo
due to impaired interactions between leukocytes and
vascular endothelial cells (21). However, the contribu-
tion of AOC3 in arthritis has not been analyzed. More-
over, the AOC3-deficient animals do not allow us to
study the contribution of the SSAO catalytic activity as
such to the adhesive functions of AOC3. Here we used
a small molecule enzyme inhibitor and AOC3-deficient
animals to unravel the importance of AOC3 and its
oxidase activity in the development of arthritis in vivo
and to test the potential of AOC3 blockade in alleviating
MATERIALS AND METHODS
Enzyme inhibitors and modeling. Synthesis of the
SSAO inhibitor BTT-2052 has been described (22). It is a trans
indane hydrazino alcohol enantiomer with the chemical for-
From the Protein Data Bank (PDB; online at www.
rcsb.org), we downloaded the crystal structures of human
AOC3 (PDB code 1us1) and other copper-containing amine
oxidases. The superposition of enzyme structures was made
with VERTAA in the BODIL modeling environment (23), and
the side-chain orientations at the ligand binding site were
studied by using the side chain rotamer library (24) incorpo-
rated in BODIL. The templates for the active conformation of
topaquinone were obtained from the crystal structures of other
copper-containing amine oxidases with bound inhibitor mole-
cules (PDB codes 1sii, 1sih, and 1spu) (25,26). The ligand
structure was energy minimized in SYBYL 7.0 (Tripos, St.
Louis, MO) with steepest descent and conjugate gradient
methods. BTT-2052 was docked manually into the ligand
binding site so that the ligand was covalently bound to the
topaquinone in a way similar to the inhibitors in the crystal
structures of other copper-containing amine oxidases (25,26).
The side-chain conformations of surrounding amino acids were
selected so that both intramolecular and intermolecular inter-
actions were energetically as favorable as possible. Some
amino acids have different conformations in the crystal struc-
ture of bovine plasma copper-containing amine oxidase (PDB
code 1tu5) (27) and human AOC3. Therefore, this structure
was also used to guide the selection of side-chain conforma-
Animals. Male Lewis rats (Harlan, Horst, The Neth-
erlands) weighing ?150 gm were used in adjuvant-induced
arthritis (AIA) studies. Wild-type and AOC3-deficient animals
(21) on a pure 129S6 background were used in the mouse
models. Age- and sex-matched animals were used in all
experiments. All animals were handled in accordance with
institutional animal care policy, and local ethics committees for
animal experiments approved the studies.
AIA in rats. Rat adjuvant arthritis was induced by
injection of 0.5 mg of dead Mycobacterium butyricum (Difco,
Detroit, MI) in 0.1 ml of liquid paraffin into the right hind
footpad. The clinical severity of the arthritis was assessed on
days 3, 7, 10, 13, 17, 20, 24, and 28 by grading each paw on a
scale of 0–4 for changes in redness and swelling, as follows: 0 ?
no changes, 1 ? paws with swelling of joints of the digits or
focal redness, 2 ? paws with mild swelling of wrist or ankle
joints; 3 ? paws with severe swelling of the entire paw, and 4 ?
paws with deformity or ankylosis. The macroscopic score was
expressed as the cumulative scores in all paws (4 paws with
scores of 0–4; maximum score 16).
Treatment was initiated on day 1, following adjuvant
injection on day 0. The rats (n ? 10 per group) were treated
either with twice daily intraperitoneal (IP) injections of BTT-
2052 in sterile water using a dosing volume of 1 ml/200 gm at
12.5 mg/kg, 25 mg/kg, or 50 mg/kg, or by mouth (also twice
daily) at 50 mg/kg. Control rats were treated with 150 mM
saline or diclofenac sodium at 2 mg/kg by mouth.
At the end of the experiment, mice were killed, and 5
uninjected ankle joints were removed from the 50 mg/kg
BTT-2052 IP-injected and saline control groups and fixed in
4% phosphate buffered formalin, decalcified, and stained.
Hematoxylin and eosin staining was used for general morphol-
ogy and Giemsa stain for mast cells and inflammatory cells.
The sections were examined for histopathologic signs of in-
flammatory arthritis, focusing mainly on the ankle joints but
also considering all smaller joints between the tarsal and
metatarsal bones, if available. Histopathologic changes were
scored by 3 independent blinded observers, assessing the
degree of inflammatory cell infiltration and neovascularization
in the synovial layers (absent ? 0, minimal ? 1, moderate ? 2,
and marked ? 3). Among all leukocytes, we estimated the
percentages of different inflammatory cells, including lympho-
cytes, macrophages, polymorphonuclear leukocytes (PMNs),
and mast cells. These cell types were identified by nuclear and
cell morphology and by staining characteristics based on
Giemsa stain. The magnitude of diffuse synovial cell layer
thickening was also scored (?5 cell layers ? 0, 5–10 cell
layers ? 1, 11–15 cell layers ? 2, and ?15 cell layers ? 3).
Cartilage and bone erosion was measured by assessing pannus
formation, formation of cavities (e.g., neovascularization
within articular cartilage), osteolytic lesions, and osteoclast
numbers, with a score of 0–3.
Anti–type II collagen antibody–induced arthritis in
mice. For the anti–type II collagen–induced arthritis model in
mice, 4 groups of animals were used: 1) wild-type controls (a
negative control group), 2) wild-type animals in which arthritis
was induced and which received no treatment, 3) wild-type
animals in which arthritis was induced and which received
SSAO inhibitor treatment, and 4) AOC3-deficient animals in
which arthritis was induced, but which did not receive any
treatment. For inducing the disease, on day 0, male 129S6 mice
(ages 8–9 weeks) were injected intravenously with 400 ?l (4
mg) of an Arthrogen-CIA Monoclonal Antibody Cocktail
(Chemicon, Temecula, CA) (28,29). In the morning of day 2,
50 ?g (200 ?l) of lipopolysaccharide (LPS) was given IP to
each mouse according to the manufacturer’s protocol to
enhance the development of the disease in this relatively
arthritis-resistant strain. To exclude any LPS-mediated effects,
PREVENTION OF LEUKOCYTE INFILTRATION BY AMINE OXIDASE INHIBITION2853
LPS was also given to the wild-type control group, which did
not receive Arthrogen. BTT-2052 was administered IP (0.625
mg in sterile water per mouse, ?25 mg/kg body weight) 2 times
a day starting in the evening of day 2.
Clinical scoring was determined by the consensus of 2
blinded investigators on day 6. In preliminary experiments,
maximal inflammation was seen around day 6 in this sponta-
neously resolving model of arthritis; therefore, day 6 was
chosen as the end point in all other studies. The severity of
arthritis was scored based on an examination of all 4 paws (29).
For each paw, points were given for swelling and redness of the
toes (0 or 5 points), knuckles/foot pad (0 or 5 points), and
wrist/ankle (0 or 5 points). Thus, every joint was scored as 0
(not affected) or 5 (affected) points, and the macroscopic score
for the animal was the cumulative sum of all joint scores
(maximum score 60; i.e., 4 paws ? 3 joints per paw ? 5 points
maximum per paw).
On day 6, mice were killed, and all 4 paws (toes,
ankle/wrist, and elbow/knee) were removed. The samples were
fixed overnight in 10% formaldehyde, decalcified with EDTA,
dehydrated, and embedded in paraffin (29). Whole joints were
sectioned at 5 ?m thickness, and every twentieth section was
stained with hematoxylin and eosin. Joint sections were scored
semiquantitatively in a blinded manner for leukocyte infiltra-
tion. The toes, wrists/ankles, and elbows/knees were scored
separately, and the numbers of inflammatory cells in the joint
space, synovium, and periarticular tissue were evaluated. The
scoring system was as follows: 0 ? no or very occasional
inflammatory cells, 1 ? moderate numbers of inflammatory
cells, and 2 ? strong or massive inflammatory infiltration.
Cloning of mouse AOC2. The full-length complemen-
tary DNA (cDNA) for mouse AOC2 was cloned from mouse
lung RNA using reverse transcriptase–polymerase chain reac-
tion (RT-PCR). Briefly, total RNA from mouse lung was
extracted using the RNeasy Mini kit (Qiagen, Hilden, Ger-
many). The RNA was reverse transcribed with Expand
Reverse Transcriptase (Roche Molecular Biochemicals,
Mannheim, Germany) as suggested by the manufacturer, and
RT-PCR was performed using the Expand Long Template
PCR System (Roche Molecular Biochemicals) according to the
instructions of the supplier. The primers used were based on
GenBank accession no. AF350446 and were as follows: 5?-
CAGTGCCAGCCATGAATCT-3? (forward) and 5?-CCT-
CAGGCCTATAAGCCTTC-3? (reverse). The resulting PCR
product of 2,293 bp was cloned into pGEM-T Easy (Promega,
Madison, WI) for sequencing and subsequently into expression
vector pcDNA3.1 (Invitrogen, Carlsbad, CA) for further
Enzyme assays. The activities of SSAOs (AOC1 [dia-
mine oxidase] , AOC2 [retina-specific amine oxidase] ,
AOC3, and lysyl oxidase [LOX] [32,33]) and flavin adenine
dinucleotide (FAD)–containing monoamine oxidases (MAOs)
A and B (34) were tested using their preferred substrates and
either fluorimetric or photometric assays, as appropriate.
Tissue lysates, purified enzymes, and transfectants were used
as the enzyme source. Recombinant human MAO-A was from
the microsomal fraction of baculovirus-infected cells (Sigma,
St. Louis, MO), purified AOC1 was from porcine kidney
(Sigma), and purified recombinant human AOC3 was from
Chinese hamster ovary (CHO) cells. For AOC1, AOC3, and
MAO assays, tissue lysates from AOC3-deficient animals and
their wild-type littermates were prepared as described else-
where (21). LOX measurements were made for the entire
aorta and lungs as described previously (35). The expression
plasmids encoding human and mouse AOC3 have been previ-
ously described (11,36). Human AOC2 was cloned from a lung
cDNA library (Elima K, Salmi M, Jalkanen S: unpublished
Fluorimetric SSAO analyses for AOC3, MAO-A, and
LOX were performed using the lysates prepared from trans-
fectants or tissues in the presence of preferred substrates and
specific inhibitors. A catalytic reaction was initiated by addi-
tion of 1 mM benzylamine (AOC3 substrate), 1 mM tyramine
(MAO-A substrate), or 10 mM cadaverine (LOX substrate),
and the Amplex red H2O2-detecting mixture (Invitrogen,
Karlsruhe, Germany). The assays were also done in the
presence of 0.5 mM clorgyline (MAO inhibitor), 200 ?M semi-
carbazide (inhibitor of SSAO), or 1 mM ?-aminopropionitrile
formed via the given enzyme reaction, the values obtained in
the presence of specific enzyme inhibitors were subtracted
from the total amount of H2O2formed. The substrate speci-
ficity assays of mouse AOC2 were performed by incubating
lysates of vector and mouse AOC2 transfectants with different
substrates (benzylamine, methylamine, ?-phenylethylamine,
tyramine, and tryptamine; 1 mM each) or without any sub-
strate in the fluorimetric assay.
Spectrophotometric assays were performed as de-
scribed elsewhere (37), by mixing the enzyme, substrates,
appropriate inhibitors, and the chromogenic solution and then
analyzing the formation of H2O2at an absorbance of 492 nm.
In these experiments, different amine oxidases (purified en-
zymes, transfectants, or tissue lysates) were preincubated with
increasing concentrations of BTT-2052 (for 15 minutes at
37°C) before the addition of the appropriate substrate and
Kivalues for inhibition of the SSAO activity of VAP-1
with the inhibitor were determined using recombinant VAP-1
expressed in CHO cells and 1 mM benzylamine as substrate, as
described previously (38). Kideterminations for total MAO
inhibition were performed using rat liver homogenates and 0.5
mM tyramine as substrate. Rat liver MAO and human liver
MAO activities were previously shown to be comparable
(results not shown). The mean ? SD Kmvalue used for VAP-1
was 90 ? 5 ?M and that for MAO was 62 ? 4 ?M.
RNA isolation and quantitative PCR. Total RNA was
isolated from cremaster muscle and Peyer’s patches using the
Ultraspec RNA isolation kit (Biotecx, Friendswood, TX) and
reverse transcribed using the iScript cDNA synthesis kit (Bio-
Rad, Hercules, CA) and DNase I digestion according to the
manufacturer’s instructions. Probes and primers for mouse
AOC2, AOC1, and ?-actin were designed using either the
Assays-by-Design (AOC1) or the Assays-on-Demand (AOC2
and ?-actin) system from Applied Biosystems (Foster City,
CA). The sequence for mouse AOC1 was derived by searching
the mouse genome with BLAST at the University of Califor-
nia, Santa Cruz web site (http://genome.ucsc.edu/) using the
human AOC1 sequence (X78212) as input. PCR reactions
were carried out as suggested by Bio-Rad using iQSupermix
and were run on an iCycler thermal cycler (Bio-Rad). The
quantitative PCR runs were performed at least twice with 2
separate cDNA samples prepared from each RNA prepara-
tion. The efficiency of the target amplification versus that of
the reference amplification was determined to be approxi-
2854 MARTTILA-ICHIHARA ET AL
mately equal by performing a validation experiment, as sug-
gested in the Applied Biosystems User Bulletin no. 2. Changes
in cycle threshold levels (?Ct) were calculated by subtracting
the average of ?-actin Ctvalues from the average of target
gene Ctvalues, and the relative expression levels of AOC2 and
AOC1 in tissues of knockout mice were shown in comparison
with those in tissues of wild-type mice.
Statistical analysis. Except where indicated otherwise,
all data are presented as the mean ? SEM. Student’s t-tests
(unpaired, 2-tailed) were used to compare the effects in
inhibitor-treated groups and in the corresponding genotypes
without the enzyme inhibitor. In the rat AIA model, the
clinical scores were statistically evaluated by analysis of vari-
ance followed by the Newman-Keuls multiple comparison test
using GraphPad Prism software, version 2.01 (GraphPad Soft-
ware, San Diego, CA). In competitive enzyme assays, the Ki,
Km, and 50% inhibition concentration (IC50) values were
computed with curve-fitting programs (GraphPad Prism, ver-
BTT-2052 binds covalently and selectively to
AOC3 and inhibits SSAO activity. We used the small
molecule SSAO inhibitor BTT-2052 (22) to study the
role of the enzymatic activity of AOC3 in arthritis in
vivo. The inhibitor is a trans indane hydrazine alcohol
enantiomer (Figure 1A). The availability of the crystal
structure of human AOC3 (39) allowed us to model the
interaction between the inhibitor and the enzyme.
AOC3 is a heart-shaped dimer having a conserved
aspartic acid catalytic base at position 386 and a unique
topaquinone cofactor (a posttranslational modification
of an intrinsic tyrosine) that is necessary for the catalytic
reaction at the active site buried deeply within the
protein and only accessible via a narrow substrate chan-
nel. Since the topaquinone is the inactive conformation
in the crystal structure, it was manually rotated into the
active conformation using the crystal structures of other
copper-containing amine oxidases with bound inhibitor
molecules as templates (25,26). The active conformation
of topaquinone is stabilized by the hydrogen bonds to
Tyr284and a water molecule that is, in turn, coordinated
to the copper ion (Figure 1B). BTT-2052 was then
docked manually into the ligand binding site so that the
Figure 1. Covalent binding of a small molecule indane hydrazino alcohol to amine oxidase, copper-
containing 3. A, Structure of BTT-2052. B, BTT-2052 (green carbon atoms) binds covalently to
topaquinone (Tpq; gray carbon atoms) and forms hydrogen bonds with Asp386and packs favorably with
the side chains of Met211, Tyr384, Phe389, and Leu469(in stereo). The topaquinone and ligand are shown
as ball-and-stick, while the surrounding amino acids and the area near the copper ion (Cu) are shown as
sticks, including 1 water molecule (w) that stabilizes the topaquinone conformation with Tyr284. The
hydrogen bonds between topaquinone–ligand and the enzyme are shown as cyan dashed lines, and the
coordination to the copper ion is shown as brown dashed lines. The figure was prepared using BODIL
(23), MOLSCRIPT (50), and Raster3D (51) software.
PREVENTION OF LEUKOCYTE INFILTRATION BY AMINE OXIDASE INHIBITION 2855
hydrazine moiety of the ligand was covalently bound to
the topaquinone in a way similar to the inhibitors in
crystal structures of other copper-containing amine oxi-
dases. As shown in Figure 1B, BTT-2052 forms hydro-
gen bonds with Asp386and exhibits good hydrophobic
packing with residues Met211, Tyr384, Phe389, and Leu469.
In enzyme assays, the inhibitor BTT-2052 effi-
ciently blocked the enzymatic activity of AOC3, but not
that of an unrelated MAO (Figure 2). Moreover, BTT-
2052 was tested in a large panel (130 targets) of in vitro
receptor binding assays and showed no significant inhi-
bition or stimulation. The structural and functional data
thus suggest that the inhibitor BTT-2052 binds co-
valently to AOC3 and blocks its catalytic activity in a
Clinical and histologic improvement of rat AIA
by SSAO inhibition. The in vivo efficacy of SSAO
inhibition in treating inflammation was studied using
different arthritis models. In rat AIA, the severity of the
disease increased in the saline-treated control group for
?3 weeks after disease induction and remained constant
thereafter. In rats treated twice daily with IP injections
of BTT-2052 at 25 and 50 mg/kg, the clinical signs of
arthritis were alleviated in a dose-dependent manner
(P ? 0.01) (Figure 3A). The clinical score in the 50
mg/kg group was reduced to that of the group treated
with diclofenac (a nonsteroidal antiinflammatory drug
used as a positive control). Importantly, when dosed
twice daily by mouth at 50 mg/kg, BTT-2052 was also
able to significantly reduce the clinical inflammation
score compared with that in saline-treated animals,
although not to the level in the diclofenac-treated con-
trol group (Figure 3B). These results show that SSAO
inhibition is highly effective at ameliorating the devel-
opment of joint inflammation in vivo in a dose-
In histologic analyses, the saline-treated group
showed that an inflammatory response dominated the
arthritis, with marked edema and widening of the intra-
articular space, including the formation of synovial sacs,
which contained neutrophil granulocytes in fibrin cloths.
Pannus formation, diffuse synovial thickening, and car-
tilage erosion along with myelosclerosis were also typi-
cal. In the BTT-2052–treated group (50 mg/kg IP), the
degree of inflammatory cell infiltration and neovascu-
larization in the synovial layers was reduced (Table 1).
Cartilage and bone erosion was also reduced, reflecting
the disease-modifying effect of SSAO inhibition (Table 1).
Figure 3. Inhibition of rat adjuvant-induced arthritis by BTT-2052.
Rats received different doses of BTT-2052 twice a day, either A,
intraperitoneally (i.p.) or B, by mouth (p.o.), and the clinical develop-
ment of the arthritis was monitored. The reduction in the mean ? SD
clinical score was significant (?? ? P ? 0.01) at the indicated time
points as compared with saline-treated animals. Diclofenac treatment
served as a positive control.
Figure 2. Selective blocking of semicarbazide-sensitive amine oxidase
(SSAO) activity by BTT-2052, an inhibitor of amine oxidase, copper-
containing 3 (AOC3; also known as vascular adhesion protein 1
[VAP-1]). Shown is the inhibition of recombinant human AOC3 and
total rat liver monoamine oxidase (tMAO) enzyme activity by BTT-
2052. The mean ? SEM Kivalue for inhibition of the SSAO activity of
recombinant human AOC3/VAP-1 with BTT-2052 was 33 ? 4 nM and
that for total rat liver MAO inhibition with BTT-2052 was 1,380 ? 110
nM. Data points are the mean ? SEM of 3 independent experiments,
each performed in duplicate.
2856 MARTTILA-ICHIHARA ET AL
On the basis of the in vitro results, BTT-2052
(Figure 2) is not a fully selective AOC3 inhibitor. To
confirm that MAO inhibition has no effect on arthritis,
a classic MAO inhibitor, pargyline, was administered IP
to the rats at a dose of 25 mg/kg once a day, which is
known to induce a complete MAO inhibition. In this
animal group, the signs of arthritis were similar in the
saline- and pargyline-treated animals (data not shown),
supporting the conclusion that the antiarthritic effect of
the AOC3 inhibitor is not due to MAO inhibition.
Alleviation of arthritis in mice by SSAO inhibi-
tion and genetic deletion of AOC3. To compare the
effects of SSAO inhibition and genetic deletion of
AOC3 in arthritis, the disease was induced in mice using
a cocktail of anti–type II collagen antibodies. Four
groups of animals were used: 1) wild-type animals with
no induction of joint inflammation (negative controls),
2) wild-type animals in which arthritis was induced and
which received no treatment (positive controls), 3) wild-
type animals in which arthritis was induced and which
were treated with the SSAO enzyme inhibitor BTT-
2052, and 4) AOC3-deficient animals in which arthritis
was induced, but which did not receive any treatment.
The clinical severity of the disease was scored blindly
and semiquantitatively on day 6 for each paw, based on
redness and swelling (Figures 4A and B). When wild-
type animals were treated with BTT-2052 starting from
day 2 of disease development, there was a significant
85% reduction in the severity of arthritis on day 6
(Figure 4C). Sixty percent of the animals were scored 0,
and only 1 animal received a score of 10. AOC3-
deficient animals also showed milder arthritis than their
wild-type littermates (Figure 4C). While 73% of the
wild-type animals had scores ?10, only 31% of the
AOC3-deficient mice had scores over this limit (P ?
0.05). Moreover, when every joint was scored separately,
there was a statistically significant reduction of clinical
inflammation both in the AOC3-deficient mice and in
the BTT-2052–treated wild-type mice (Figure 4D).
In this mouse model of acute arthritis, there was
marked infiltration of PMNs in most joints of the
untreated wild-type mice (Figures 5A–D). The number
of infiltrating PMNs was significantly reduced in animals
treated with the SSAO inhibitor BTT-2052, and the
reduction was most striking in the small joints (Figure
5E). AOC3-deficient animals in which arthritis was
induced also had significantly fewer infiltrating leuko-
cytes than their wild-type arthritic controls (Figure 5E).
The 2 different arthritis models thus show that genetic
deletion of AOC3 or blockade of the AOC3 enzyme
activity by a small molecule SSAO inhibitor improves
the clinical and histologic outcome of arthritis in vivo.
SSAO inhibitor BTT-2052 blocks several amine
oxidases. Direct comparison of the arthritis-suppressing
potential of AOC3 targeting surprisingly revealed that
acute SSAO blockade with the enzyme inhibitor caused
more robust changes than deletion of the AOC3 gene
(Figures 4 and 5). Since there are multiple copper-
containing amine oxidases in mice and humans (30–34),
these data suggest that the SSAO inhibitor may block
other oxidases in addition to AOC3.
Comparison of wild-type and AOC3-deficient
Histopathologic analysis of BTT-2052 in rat adjuvant-induced arthritis*
Inflammatory cells, %‡
(range 0–3)¶PMNs MacrophagesLymphocytes
* PMNs ? polymorphonuclear leukocytes.
† Semiquantitative analysis of the degree of inflammatory cell infiltration and neovascularization in synovium.
‡ Percentages of different leukocyte types among all leukocytes. No mast cells were found.
§ Semiquantitative scoring of diffuse synovial layer thickening.
¶ Semiquantitative scoring of pannus formation, formation of cavities, osteolytic lesions, and osteoclast numbers.
PREVENTION OF LEUKOCYTE INFILTRATION BY AMINE OXIDASE INHIBITION 2857
animals showed that the SSAO activity was almost
completely abolished in normally AOC3-rich tissues in
the AOC3-knockout mice (Figure 6A). There were no
significant changes in the enzymatic activity and/or
messenger RNA (mRNA) synthesis of copper-
containing AOC1 (diamine oxidase) or FAD-containing
MAOs A and B between the wild-type and AOC3-
deficient animals (Figures 6A and B). Interestingly, we
found a compensatory increase in the synthesis of AOC2
mRNA (also known as retina-specific amine oxidase ,
although it is expressed in multiple tissues [Elima K,
Salmi M, Jalkanen S: unpublished observations]) in
AOC3-deficient animals (Figure 6B).
In competitive enzyme assays, BTT-2052 was
shown to efficiently and dose-dependently inhibit mouse
and human AOC3, with IC50values in the nanomolar
Figure 4. Alleviation of clinical arthritis by SSAO inhibition and AOC3 deletion. Arthritis was induced in mice using anti–type
II collagen antibodies. Representative photographs of A, normal and B, inflamed front paws are shown. Note the redness and
swelling at the wrist and knuckles in the inflamed paw. Clinical severity of arthritis was scored for C, each animal (red dot
represents the mean of each group) and D, each paw (mean and SEM). ? ? P ? 0.05; ?? ? P ? 0.01.?/?? wild type;?/??
AOC3 deficient; SSAOinh ? SSAO enzyme inhibitor BTT-2052 (see Figure 2 for other definitions).
Figure 5. Histologic amelioration of synovitis in the absence of AOC3 oxidase activity. Representative photomicrographs of
A, normal (score 0), B, mildly inflamed (score 1), and C and D, strongly inflamed (score 2) wrist joints are shown. Both mildly
and strongly inflamed wrist joints show migration of granulocytes into affected tissue (original magnification ? 100 in A–C; ?
400 in D). E, The mean and SEM inflammatory cell influx was scored for each joint of every animal in each group. The animals
were wild type (AOC3?/?) or AOC3 deficient (AOC3?/?) and were either untreated or treated with 50 mg/kg BTT-2052
(SSAOin). ?? ? P ? 0.01. See Figure 2 for other definitions.
2858 MARTTILA-ICHIHARA ET AL
range (Figures 6C and D). It did not affect the activity of
mouse, human, or rat MAO (Figures 2 and 6C and D).
In contrast, it dose-dependently inhibited the activity of
purified AOC1 (from pig kidney). Using transient
transfections and multiple potential AOC substrates,
we found that ?-phenylethylamine, tyramine, and
tryptamine were potential substrates for mouse AOC2
(Figure 6E). Since the enzymatic activity of mouse
Figure 6. Inhibition of amine oxidases AOC1 and AOC3 by SSAO inhibitior BTT-2052. A, The mean and SEM enzymatic activity of different amine
oxidases in wild-type (AOC3?/?) and AOC3-deficient (AOC3?/?) animals was measured fluorimetrically from the indicated tissue lysates using the
preferred substrates tyramine (Tyram), putrescine (Putresc), methylamine (MA), and cadaverine (Cadav). LOX ? lysyl oxidase. B, The mean
mRNA expression of AOC1 (from Peyer’s patches) and AOC2 (from cremaster muscle) in wild-type and AOC3-deficient animals was determined
using quantitative polymerase chain reaction. C, The effect of BTT-2052 compound on the enzymatic activity of different amine oxidases was
analyzed using photometric assays and purified enzymes or tissue and transfectant lysates. mAOC3 ? mouse AOC3 and hAOC3 ? human AOC3
(mAOC3, hAOC3, and hAOC2 were all from HEK 293 transfectants); rAOC3 ? recombinant human AOC3 purified from Chinese hamster ovary
cell transfectants; AOC1 ? purified porcine AOC1; mSSAO ? mouse SSAO from fat; mMAO ? mouse MAO activity from liver; MAO-A ?
recombinant human MAO-A. D, The mean and SEM 50% inhibition concentration (IC50) values for BTT-2052 against different amine oxidases were
determined (note the different scale of the y-axis) (n ? 4–5 experiments for all measurements). E, Substrate specificity of mouse AOC2. The mean
and SEM activity (n ? 6 experiments) was determined from vector- or mouse AOC2–transfected HEK 293 cells by incubating the lysates with the
indicated amines using the fluorimetric assay. PEA ? ?-phenylethylamine (see Figure 2 for other definitions).
PREVENTION OF LEUKOCYTE INFILTRATION BY AMINE OXIDASE INHIBITION 2859
AOC2 was low in our expression system, we were not
able to directly assess the inhibitory potential of BTT-
2052 on it. Nevertheless, since the preferential order of
amine substrates is exactly the same with human and
mouse AOC2, we were able to test the effect of BTT-
2052 on the human enzyme, which gave higher enzy-
matic activity. These data clearly show that BTT-2052
also inhibits AOC2, although less potently than AOC1.
Therefore, either AOC1 or AOC2 or both may also be
involved in regulating leukocyte–endothelial contacts
Here we have shown for the first time that AOC3
is important for the pathogenesis of arthritis in vivo.
When the SSAO activity of AOC3 was blocked by a
small molecule inhibitor, the clinical and histologic signs
of arthritis were reduced in 2 different models. Since the
SSAO inhibitor also blocked arthritis when given by
mouth, it could be useful for clinical administration.
The inhibitory effect of anti-AOC3 antibodies on
inflammation has been documented in many animal
studies and was therefore not analyzed in this study. In
contrast, very little is known about the importance of the
oxidase activity of AOC3 in supporting leukocyte traf-
ficking in vivo. In in vitro assays, the adhesive function of
AOC3 can be blocked either by mAb that do not inhibit
the enzymatic activity or by SSAO inhibitors (16,17,20).
These data have led to the current working model that
leukocytes first interact with AOC3 via adhesive
epitopes that are detected by the anti-AOC3 mAb, and
then via a leukocyte surface–expressed substrate (such
as galactosamine or a lysine-containing peptide) that can
penetrate through a narrow substrate channel into the
catalytic site, buried deep inside the enzyme, to initiate
the SSAO reaction. If either of these steps is missing,
AOC3 is functionally inactive. AOC3-deficient animals
cannot be used to verify this hypothesis in vivo, since
both the antibody epitopes and SSAO activity are miss-
ing from those animals. Our current data show that
SSAO enzyme activity is indeed needed for inflamma-
tion in vivo. The enzyme reaction results in the forma-
tion of an aldehyde, ammonium, and H2O2. All these
bioactive products could be involved in the modulation
of leukocyte extravasation. In particular, H2O2is known
to alter the expression of other adhesion molecules
(P-selectin, vascular cell adhesion molecule 1), chemo-
kine receptors, and matrix metalloproteinases (40–43)
and may thus modulate leukocyte extravasation by sig-
AOC3 blockade caused a partial alleviation of
joint inflammation. The partial, rather than complete,
effect is typical of blocking of most other adhesion
molecules as well. This is inherently related to the nature
of the vital extravasation cascade. Thus, during elicita-
tion of a vigorous inflammation, such as that seen in the
large joints of arthritic mice, the inflammatory stimuli
may be so overwhelming that all, or at least multiple,
parallel extravasation pathways are maximally activated.
In this case, the redundancy of the extravasation system
will allow compensatory, non–SSAO-mediated, path-
ways to be used to a larger extent to ensure proper
defense reactions. In any case, the utility of SSAO
inhibitors in alleviating inflammation when administered
by mouth supports the feasibility of this approach in
controlling arthritis in vivo.
The SSAO inhibition surprisingly caused larger
effects on leukocyte–endothelial interactions and in-
flammatory disease than did deletion of the AOC3 gene.
BTT-2052 was derived from a chemical screen designed
to find new SSAO inhibitors (22). It is a water-soluble
carbocyclic hydrazine compound that has suitable phys-
icochemical properties to be used in in vivo experiments,
and hence, it is superior to the prototype SSAO inhibi-
tors semicarbazide and hydroxylamine. The inhibitor did
not affect the hematologic parameters (data not shown)
and did not cause any obvious side effects in the current
arthritis models or in preclinical tests at concentrations
used in vivo. The compound is a potent inhibitor of
AOC3 and did not show significant activity against
MAO or multiple unrelated molecules including many
key receptors, kinases, and other enzymes. However, our
data showed that BTT-2052 also inhibits AOC1 and
AOC2. These 2 enzymes are expressed in endothelial
cells in many tissues (ref. 44, and Elima K, Salmi M,
Jalkanen S: unpublished observations) and could medi-
ate SSAO-catalyzed reactions involved in leukocyte ad-
hesion in a manner similar to that described for AOC3.
It should be emphasized that currently, no inhib-
itors specific to a given SSAO species are available,
making it impossible to directly demonstrate the involve-
ment of the other SSAO molecules in leukocyte extrav-
asation. The stress-induced glucocorticoid effects may
have potentiated the antiinflammatory effects in the
SSAO inhibitor–treated group as compared with the
control and AOC3-deficient groups in our experimental
setting, since we did not include a separate control group
that received daily vehicle injections (to reduce the
numbers of animals needed). However, we have seen in
another model that the SSAO inhibitor indeed has
greater effects on the inflammation than does ablation
of the AOC3 gene. Using real-time imaging of
leukocyte–endothelial cell interactions in inflamed cre-
2860MARTTILA-ICHIHARA ET AL
master vasculature, we observed that the inhibition of
leukocyte extravasation was less profound in AOC3-null
animals than in wild-type animals treated with the SSAO
inhibitor (Marttila-Ichihara F, Salmi M: unpublished
observations). Moreover, leukocyte extravasation was
diminished more in the AOC3-deficient mice treated
with the SSAO inhibitor than in AOC3-deficient animals
treated with the vehicle. Finally, we also cannot exclude
the possibility that the SSAO inhibitor used in the
current study, or in the cremaster model, might also
inhibit some other molecule involved in inflammation.
Nevertheless, our current data clearly show that block-
ade of the SSAO activity can profoundly alleviate in-
flammatory reactions in vivo. Findings of the very recent
studies using different SSAO inhibitors and different
models of inflammation fully support our conclusions
AOC3 has several benefits as a potential target
for antiadhesive therapy. It is normally virtually absent
from the luminal surface of uninflamed endothelium.
Upon inflammation, AOC3 is rapidly translocated from
intracellular storage vesicles to the luminal surface, both
in mice and in humans (47,48). Since only surface-
expressed enzyme is available for binding neutralizing
anti-AOC3 antibodies, and since it is enzymatically
active only at this location (37), blocking of its function
should have minimal effects on the function of the
normal immune surveillance systems. This is, in fact,
consistent with the observed phenotype of AOC3-
deficient animals (21). In the absence of AOC3, mice are
microscopically and macroscopically healthy and have
only minor defects in leukocyte trafficking to the gut
under normal conditions. Moreover, most adhesion mol-
ecules are currently being targeted by humanized anti-
bodies, which have their inherent limitations in clinical
use. This is primarily due to the fact that many other
adhesion molecules are difficult to target with small
molecular compounds. In contrast, the enzymatic nature
and available crystal structure of AOC3 greatly facilitate
When used in in vitro adhesion assays, anti-
AOC3 mAb inhibit the binding of leukocytes to human
vessels in several inflammatory disorders, including ar-
thritis (49). The good in vivo efficacy of acute adminis-
tration of an SSAO inhibitor in alleviating arthritis in
rodents therefore suggests that such inhibitors could be
useful in the clinical setting. The possibility of targeting
adhesive cell-surface enzymes with a small molecule
compound offers new venues for antiadhesive therapy
that can circumvent many of the difficulties encountered
when using neutralizing mAb.
We thank Prof. Mark Johnson for providing the facil-
ities for the modeling studies and Anne Sovikoski-Georgieva
for secretarial help.
1. Von Andrian UH, Mackay CR. T-cell function and migration. Two
sides of the same coin. N Engl J Med 2000;343:1020–34.
2. Ley K, Kansas GS. Selectins in T-cell recruitment to non-lymphoid
tissues and sites of inflammation. Nat Rev Immunol 2004;4:
3. Rosen SD. Ligands for L-selectin: homing, inflammation, and
beyond. Annu Rev Immunol 2004;22:129–56.
4. Kunkel EJ, Butcher EC. Chemokines and the tissue-specific
migration of lymphocytes. Immunity 2002;16:1–4.
5. Miyasaka M, Tanaka T. Lymphocyte trafficking across high endo-
thelial venules: dogmas and enigmas. Nat Rev Immunol 2004;4:
6. Muller WA. Leukocyte-endothelial-cell interactions in leukocyte
transmigration and the inflammatory response. Trends Immunol
7. Pribila JT, Quale AC, Mueller KL, Shimizu Y. Integrins and T
cell-mediated immunity. Annu Rev Immunol 2004;22:157–80.
8. Dejana E. Endothelial cell-cell junctions: happy together. Nat Rev
Mol Cell Biol 2004;5:261–70.
9. Salmi M, Jalkanen S. VAP-1: an adhesin and an enzyme. Trends
10. Salmi M, Jalkanen S. Cell-surface enzymes in control of leukocyte
trafficking. Nat Rev Immunol 2005;5:760–71.
11. Smith DJ, Salmi M, Bono P, Hellman J, Leu T, Jalkanen S.
Cloning of vascular adhesion protein-1 reveals a novel multifunc-
tional adhesion molecule. J Exp Med 1998;188:17–27.
12. Klinman JP, Mu D. Quinoenzymes in biology. Annu Rev Biochem
13. Jalkanen S, Salmi M. Cell surface monoamine oxidases: enzymes
in search of a function. EMBO J 2001;20:3893–901.
14. Salmi M, Jalkanen S. A 90-kilodalton endothelial cell molecule
mediating lymphocyte binding in humans. Science 1992;257:1407–9.
15. Salmi M, Jalkanen S. Human vascular adhesion protein-1 (VAP-1)
is a unique sialoglycoprotein that mediates carbohydrate-depen-
dent binding of lymphocytes to endothelial cells. J Exp Med
16. Lalor PF, Edwards S, McNab G, Salmi M, Jalkanen S, Adams DH.
Vascular adhesion protein-1 mediates adhesion and transmigra-
tion of lymphocytes on human hepatic endothelial cells. J Immu-
17. Salmi M, Yegutkin G, Lehvonen R, Koskinen K, Salminen T,
Jalkanen S. A cell surface amine oxidase directly controls lympho-
cyte migration. Immunity 2001;14:265–76.
18. Bonder C, Swain MG, Zbytnuik LD, Norman MU, Yamanouchi J,
Santamaria P, et al. Rules of recruitment of trafficking Th1 and
Th2 cells in inflamed liver. Immunity 2005;23:153–63.
19. Tohka S, Laukkanen ML, Jalkanen S, Salmi M. Vascular adhesion
protein 1 (VAP-1) functions as a molecular brake during granu-
locyte rolling and mediates their recruitment in vivo. FASEB J
20. Koskinen K, Vainio PJ, Smith DJ, Pihlavisto M, Yla-Herttuala S,
Jalkanen S, et al. Granulocyte transmigration through endothe-
lium is regulated by the oxidase activity of vascular adhesion
protein-1 (VAP-1). Blood 2004;103:3388–95.
21. Stolen CM, Marttila-Ichihara F, Koskinen K, Yegutkin GG, Turja
R, Bono P, et al. Absence of the endothelial oxidase AOC3 leads
to abnormal leukocyte traffic in vivo. Immunity 2005;22:105–15.
22. Smith DJ, Fulop F, Pihlavisto M, Lazar L, Alaranta S, Vainio PJ,
et al. Carbocyclic hydrazino inhibitors of copper-containing amine
PREVENTION OF LEUKOCYTE INFILTRATION BY AMINE OXIDASE INHIBITION 2861
oxidases. Int Pat Appl 2003; Publ. No.: WO 03/006003 A1; Chem
Abstr. 138, 57890.
23. Lehtonen JV, Still DJ, Rantanen VV, Ekholm J, Bjorklund D,
Iftikhar Z, et al. BODIL: a molecular modeling environment for
structure-function analysis and drug design. J Comput Aided Mol
24. Lovell SC, Word JM, Richardson JS, Richardson DC. The penul-
timate rotamer library. Proteins 2000;40:389–408.
25. O’Connell KM, Langley DB, Shepard EM, Duff AP, Jeon HB, Sun
G, et al. Differential inhibition of six copper amine oxidases by a
family of 4-(aryloxy)-2-butynamines: evidence for a new mode of
inactivation. Biochemistry 2004;43:10965–78.
26. Wilmot CM, Murray JM, Alton G, Parsons MR, Convery MA,
Blakeley V, et al. Catalytic mechanism of the quinoenzyme amine
oxidase from Escherichia coli: exploring the reductive half-reac-
tion. Biochemistry 1997;36:1608–20.
27. Lunelli M, Di Paolo ML, Biadene M, Calderone V, Battistutta R,
Scarpa M, et al. Crystal structure of amine oxidase from bovine
serum. J Mol Biol 2005;346:991–1004.
28. Terato K, Hasty KA, Reife RA, Cremer MA, Kang AH, Stuart
JM. Induction of arthritis with monoclonal antibodies to collagen.
J Immunol 1992;148:2103–8.
29. Nandakumar KS, Svensson L, Holmdahl R. Collagen type II-
specific monoclonal antibody-induced arthritis in mice: description
of the disease and the influence of age, sex, and genes. Am J
30. Chassande O, Renard S, Barbry P, Lazdunski M. The human gene
for diamine oxidase, an amiloride binding protein. Molecular
cloning, sequencing, and characterization of the promoter. J Biol
31. Imamura Y, Noda S, Mashima Y, Kudoh J, Oguchi Y, Shimizu N.
Human retina-specific amine oxidase: genomic structure of the
gene (AOC2), alternatively spliced variant, and mRNA expression
in retina. Genomics 1998;51:293–8.
32. Kagan HM, Li W. Lysyl oxidase: properties, specificity, and
biological roles inside and outside of the cell. J Cell Biochem
33. Csiszar K. Lysyl oxidases: a novel multifunctional amine oxidase
family. Prog Nucleic Acid Res Mol Biol 2001;70:1–32.
34. Nagatsu T. Progress in monoamine oxidase (MAO) research in
relation to genetic engineering. Neurotoxicology 2004;25:11–20.
35. Palamakumbura AH, Trackman PC. A fluorometric assay for
detection of lysyl oxidase enzyme activity in biological samples.
Anal Biochem 2002;300:245–51.
36. Bono P, Salmi M, Smith DJ, Jalkanen S. Cloning and character-
ization of mouse vascular adhesion protein-1 reveals a novel
molecule with enzymatic activity. J Immunol 1998;160:5563–71.
37. Yegutkin GG, Salminen T, Koskinen K, Kurtis C, McPherson MJ,
Jalkanen S, et al. A peptide inhibitor of vascular adhesion pro-
tein-1 (VAP-1) blocks leukocyte-endothelium interactions under
shear stress. Eur J Immunol 2004;34:2276–85.
38. Holt A, Sharman DF, Baker GB, Palcic MM. A continuous
spectrophotometric assay for monoamine oxidase and related
enzymes in tissue homogenates. Anal Biochem 1997;244:384–92.
39. Airenne TT, Nymalm Y, Kidron H, Smith DJ, Pihlavisto M, Salmi
M, et al. Crystal structure of the human vascular adhesion
protein-1: unique structural features with functional implications.
Protein Sci 2005;14:1964–74.
40. Johnston B, Kanwar S, Kubes P. Hydrogen peroxide induces
leukocyte rolling: modulation by endogenous antioxidant mecha-
nisms including NO. Am J Physiol 1996;271:H614–21.
41. Saccani A, Saccani S, Orlando S, Sironi M, Bernasconi S, Ghezzi
P, et al. Redox regulation of chemokine receptor expression. Proc
Natl Acad Sci U S A 2000;97:2761–6.
42. Yoon SO, Park SJ, Yoon SY, Yun CH, Chung AS. Sustained
production of H(2)O(2) activates pro-matrix metalloproteinase-2
through receptor tyrosine kinases/phosphatidylinositol 3-kinase/
NF-? B pathway. J Biol Chem 2002;277:30271–82.
43. Willam C, Schindler R, Frei U, Eckardt KU. Increases in oxygen
tension stimulate expression of ICAM-1 and VCAM-1 on human
endothelial cells. Am J Physiol 1999;276:H2044–52.
44. Baenziger NL, Mack P, Jong YJ, Dalemar LR, Perez N, Lindberg
C, et al. An environmentally regulated receptor for diamine
oxidase modulates human endothelial cell/fibroblast histamine
degradative uptake. J Biol Chem 1994;269:14892–8.
45. Salter-Cid LM, Wang E, O’Rourke AM, Miller A, Gao H, Huang
L, et al. Anti-inflammatory effects of inhibiting the amine oxidase
activity of semicarbazide-sensitive amine oxidase. J Pharmacol
Exp Ther 2005;315:553–62.
46. Xu HL, Salter-Cid L, Linnik M, Wang E, Paisansathan C, Pelli-
grino D. Vascular adhesion protein-1 plays an important role in
post-ischemic inflammation and neuropathology in diabetic, estro-
gen-treated ovariectomized female rats subjected to transient
forebrain ischemia. J Pharmacol Exp Ther 2005;317:19–29.
47. Jaakkola K, Nikula T, Holopainen R, Vahasilta T, Matikainen
MT, Laukkanen ML, et al. In vivo detection of vascular adhesion
protein-1 in experimental inflammation. Am J Pathol 2000;157:
48. Vainio PJ, Kortekangas-Savolainen O, Mikkola JH, Jaakkola K,
Kalimo K, Jalkanen S, et al. Safety of blocking vascular adhesion
protein-1 in patients with contact dermatitis. Basic Clin Pharmacol
49. Salmi M, Rajala P, Jalkanen S. Homing of mucosal leukocytes to
joints. Distinct endothelial ligands in synovium mediate leukocyte-
subtype specific adhesion. J Clin Invest 1997;99:2165–72.
50. Kraulis J. MOLSCRIPT: a program to produce both detailed and
schematic plots of protein structures. J Appl Crystallogr 1991;24:
51. Merritt E, Bacon D. Raster3D: photorealistic molecular graphics.
Methods Enzymol 1997;277:505–24.
2862MARTTILA-ICHIHARA ET AL