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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Developmental regulation of the adhesive and enzymatic activity of vascular
adhesion protein-1 (VAP-1) in humans
Marko Salmi and Sirpa Jalkanen
Vascular adhesion protein-1 (VAP-1) is a
homodimeric glycoprotein that belongs
to a unique subgroup of cell-surface–
expressed oxidases. In adults, endothelial VAP-1 supports leukocyte rolling, firm
adhesion, and transmigration in both enzyme activity–dependent and enzyme activity–independent manner. Here we studied the induction and function of VAP-1
during human ontogeny. We show that
VAP-1 is already found in the smooth
muscle at embryonic week 7. There are
marked time-dependent switches in VAP-1
expression in the sinusoids of the liver, in
the peritubular capillaries of the kidney,
in the capillaries of the heart, and in the
venules in the lamina propria of the gut.
Fetal VAP-1 is dimerized, and it is enzymatically active. VAP-1 in fetal-type
venules is able to bind cord blood lymphocytes. Also, adenovirally transfected
VAP-1 on human umbilical vein endothelial cells is involved in rolling and firm
adhesion of cord blood lymphocytes un-
der conditions of physiologic shear
stress. We conclude that VAP-1 is synthesized from early on in human vessels and
it is functionally intact already before
birth. Thus, VAP-1 may contribute critically to the oxidase activities in utero,
and prove important for lymphocyte trafficking during human ontogeny. (Blood.
2006;108:1555-1561)
© 2006 by The American Society of Hematology
Introduction
Leukocyte trafficking between the blood and lymphoid organs is
vital for the immune defense. In adults, the extravasation is
governed by a multistep adhesion cascade, which involves sequential interactions between endothelial adhesion molecules and their
receptors on leukocytes.1,2 One of the endothelial molecules
involved in this process is vascular adhesion protein-1 (VAP-1). It
is a homodimeric 180-kDa glycoprotein expressed on vascular
endothelium.3-5 In vitro binding assays have shown that VAP-1 is
needed for leukocyte rolling, firm adhesion, and transmigration
during the extravasation cascade in adults.6-8 Animal work using
neutralizing anti–VAP-1 antibodies and VAP-1–deficient mice has
confirmed its importance in leukocyte trafficking during the
physiologic and pathologic conditions in vivo.9-13
VAP-1 belongs to a group of enzymes called semicarbazide
sensitive amine oxidases (SSAOs; Enzyme Commission [EC]
number 1.4.3.6).14,15 SSAOs catalyze the general reaction R-CH2NH2 ⫹ H2O 3 R-CH2-CHO ⫹ H2O2 ⫹ NH3.16,17 In man and mice
3 SSAOs, VAP-1, diamine oxidase, and retina-specific amine
oxidase, are known.14,18,19 These amine oxidases are notably
distinct from monoamine oxidases A and B in terms of subcellular
localization, substrates, inhibitors, cofactors, and function. In fact,
leukocyte-endothelial adhesion was the first physiologic function
ascribed to SSAOs despite intense research of more than 50 years.
Interestingly, this cellular interaction apparently takes advantage of
the catalytic nature of VAP-1, since the transient but covalent bond
between the enzyme (endothelial VAP-1) and substrate (a surfaceexpressed amine on leukocyte) is important for leukocyte–
endothelial cell interactions under conditions of shear flow.6,15 In
addition to endothelium, VAP-1 is expressed in adipocytes, in
dendritic cells of germinal centers, and in smooth muscle cells, but
not in other types of muscle, in adults.4,20 In adipocytes it
contributes to the glucose metabolism and fat cell differentiation,21-25 but on other cell types its functions remain unknown. In
addition to the transmembrane VAP-1, a soluble form of the
molecule circulates in the blood of adults, and it may modulate the
adhesive activity of surface-bound VAP-1.26,27
The mechanisms guiding lymphocyte trafficking during fetal
development have remained practically unknown. Moreover, nothing is known from the expression or function of any SSAO
molecule before birth. Therefore, we studied the induction and
function of the membrane-bound and soluble form of VAP-1 during
the human ontogeny.
From the National Public Health Institute, Turku; and MediCity Research
Laboratory, University of Turku, Finland.
Reprints: Marko Salmi, MediCity Research Laboratory, Tykistökatu 6A, 20520
Turku, Finland; e-mail: [email protected].
Submitted November 22, 2005; accepted March 12, 2006. Prepublished online as
Blood First Edition Paper, March 23, 2006; DOI 10.1182/blood-2005-11-4599.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
Supported by the Finnish Academy and Sigrid Juselius Foundation.
© 2006 by The American Society of Hematology
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
Materials and methods
Fetal tissues and cells
Human tissue samples were collected from aborted fetuses, from autopsies,
and from surgical operations. The 7- and 9-week-old embryos were whole
mounts. From later time points (11 weeks [n ⫽ 3]), 13 weeks [n ⫽ 2], 15
weeks [n ⫽ 3], 18 weeks [n ⫽ 4], 21 weeks [n ⫽ 4], 23 weeks [n ⫽ 2], 33
weeks [n ⫽ 1], and 40 weeks [n ⫽ 6]), multiple organs were collected.
These included the following lymphatic tissues: spleen, peripheral lymph
nodes (PLNs), small and large bowel, thymus, and bone marrow. In
addition, the heart, lung, liver, kidney, adrenal, thyroid, pancreas, ovary,
skin, brain, umbilical cord and placenta were obtained. The same organs
were also obtained from 2-month-old (n ⫽ 2), 2-year-old (n ⫽ 2), and
7-year-old (n ⫽ 1) children, and from adults (n ⫽ 4). The low number of
samples and irregular and missing time points were due to the difficulty of
1555
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1556
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
SALMI and JALKANEN
obtaining fetal and childhood samples. Our fetal samples younger than 20
weeks were from spontaneous or induced abortions performed by the
treating obstetricians according to the Finnish law at the Turku University
Central Hospital. Other fetal samples were from stillbirths. The samples
from children were obtained from deceased children who underwent
autopsy due to clinical reasons. All samples were macroscopically and
microscopically normal. Both snap-frozen samples and formalin-fixed,
paraffin-embedded samples were prepared, if possible. Not all tissues and
organs were available from every individual, but all the results reported
were ascertained in a minimum of 2 samples from different individuals of
the same age.
Cord blood and adult peripheral blood lymphocytes (PBLs) were
isolated using density gradient centrifugation over Ficoll. Serum from cord
and adult blood were collected according to standard procedures, aliquoted
and stored immediately at ⫺70 °C.
The study protocol was approved by the local Ethics Committee and by
the National Board of Medicolegal Affairs in Finland.
Immunohistochemistry
Monoclonal antibodies (mAbs) against human VAP-1 (2D10 and TK8-18)
and against mouse VAP-1 (TK10-79) have been described.26,28 2D10 and
TK8-14 are function-blocking mAbs that only work with frozen sections,
whereas TK10-79, which is cross-reactive with human VAP-1, works with
paraffin sections. Anti-CD31 mAb was from Dako (Glostrup, Denmark). As
negative controls, mAbs (MECA-367 against mouse MAdCAM-1 and 3G6
against chicken T cells) not reactive with human antigens, and a binding,
but not adhesion-blocking mAb, HB151, against human leukocyte antigen
(HLA) class I were used.8,29,30
Staining of acetone fixed frozen sections was done using indirect
immunoperoxidase staining as described.3 Staining of paraffin sections was
done using microwave treatment in a citrate buffer for antigen retrieval and
subsequent staining with the avidin-peroxidase method using Vectastain
kits (Vector Laboratories, Burlingame, CA) as described.28 All samples
were stained for VAP-1, CD31, and negative control. Images were obtained
using an Olympus BX60 microscope equipped with a UPlanF1 20⫻/0.50
numeric aperture [NA] Ph1 or UPlanF1 40⫻/0.75 NA Ph2 objective.
Images were acquired using a ColorView 12 camera (Olympus Soft
Imaging Solutions, Münster, Germany).
Biochemical analyses
The molecular weight of VAP-1 in tissues was determined by immunoblotting. Tissue fragments were lysed in 1% NP-40–containing buffer, resolved
in SDS-PAGE electrophoresis, and subjected to immunoblotting using ECL
detection exactly as described earlier.5
Depletion of VAP-1 from the lysates or serum samples was performed
by 3 sequential incubations with cyanogen-bromide–activated Sepharose
4CL beads to which anti–VAP-1 or control mAb were coupled according to
a previously described protocol.27
Adhesion assays
The Stamper-Woodruff assay was done as reported.31 In brief, frozen
sections of PLNs, intestines, and livers were pretreated with anti–VAP-1
and control mAbs (100 ␮g/mL), and lymphocytes in suspension were
allowed to bind for 30 minutes under constant rotation at 7°C. The results
are expressed as a percentage of maximal binding, which is defined by the
number of lymphocytes adherent per vessel in control mAb–treated
sections. At least 100 vessels were analyzed for each experimental
condition in each experiment.
To mimic physiologic blood flow, adhesion assays were also performed
using in vitro capillary flow assays with laminar shear stress using a
previously described protocol.8,32 In brief, human umbilical vein endothelial cells (HUVECs) were infected with adenoviral construct encoding for
VAP-1, grown into confluency, and stimulated with tumor necrosis factor-␣
(TNF-␣; 100 U/mL for 4 hours). The monolayer was then treated with
anti–VAP-1 and control mAbs (10 ␮g/ml) or with an SSAO inhibitor (5 ␮M
hydroxylamine) for 20 minutes. Peripheral blood mononuclear cells
(PBMCs) were suspended in RPMI1640 containing 0.1% bovine serum
albumin (BSA) and 5 ␮M histamine and drawn over the endothelial
monolayer using a computer-controlled pump and laminar shear stress of
1.0 dyn/cm2 at room temperature. For analysis of rolling and firm adhesion,
the cells were allowed to interact for 5 minutes under the shear. For the
transmigration assay, the cells were perfused for 10 minutes. Thereafter, the
cells were allowed to transmigrate for additional 20 minutes in the presence
of continuous flow (without lymphocytes in the flow buffer). After an intial
equilibrization period, the interaction of leukocytes with the endothelial
cells was observed for 5 minutes (10 predetermined fields, 30 seconds each
in rolling/firm adhesion assays and 15 fields in the transmigration assays)
under an Olympus IX70 inverted camera (Olympus Optical, Hamburg,
Germany) equipped with a Hamamatsu C5405-01 charge-coupled device
(CCD) camera (Hamamatsu Photonics, Hamamatsu, Japan). Objectives
used included an HMC 10⫻ Plan UIS (NA 0.25; Modulation Optics,
Greenvale, NY) and a UPlan FL 10⫻/0.3 NA (Olympus Optical), along
with an IX-LWVCD (NA 0.55) condenser (Olympus Optical). Imaging
medium was RPMI 1640 containing 0.1% BSA. Image acquisition software
was AnalySIS (Olympus Soft Imaging Systems, Münster, Germany).
Leukocyte-endothelial interactions were then determined offline, as
described.8,32 The number of interacting cells observed in the presence of
control treatment (anti–HLA-I mAb or vehicle, as appropriate) was defined
as 100%. In the rolling/adhesion assays the mean absolute number of
analyzed rolling cells per 1 experiment (n ⫽ 5 independent experiments
using different HUVECs and cord blood cells) was 74 (corresponding to 24
cells/mm2), and that of firmly adherent cells 646 (corresponding to 210
cells/mm2) in the control capillaries. In the transmigration assays, on
average 618 adherent and 62 transmigrated cells (corresponding to 134 and
13 cells/mm2, respectively) were scored in each control capillaries (ie, on
average 10% of the interacting cells transmigrated in the control capillaries).
Enzymatic assays
The oxidative activity of VAP-1 was measured using a fluoropolarimetric
assay, which measures the hydrogen peroxide production in the SSAOcatalyzed reaction, as described.6
Soluble VAP-1 ELISA
The amount of soluble VAP-1 in serum samples was determined using a
sandwich enzyme-linked immunosorbent assay (ELISA), exactly as
described.26
Statistical analyses
The effect of an anti–VAP-1 antibody or SSAO enzyme inhibitor treatment
was compared with the control samples (treated with a control mAb or
vehichle, as appropriate) using Student t test. Significance was set at a P
value less than .05.
Results
Induction of VAP-1 in specific vascular beds early during fetal
development in humans
Faint expression of VAP-1 was seen already in our earliest
human samples from embryonic week 7. At that stage, VAP-1
expression was confined to the smooth muscle layer of the aortic
and gut wall (Figure 1). Thereafter, VAP-1 synthesis appeared in
smooth muscle and fat cells in different organs. Notably,
skeletal or cardiac muscle cells lacked VAP-1 at all developmental stages.
In certain organs developmental switches in VAP-1 synthesis
were evident. In the liver, the central vein and smooth muscle
cells in the portal tract were faintly VAP-1 positive from 11
weeks onwards. However, the sinusoidal endothelium first
showed VAP-1 expression at week 18, and the level of VAP-1
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BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
VAP-1 DURING HUMAN ONTOGENY
1557
tory pathologies,4,33-35 become VAP-1 positive gradually and
during different time points of fetal development.
Expression of VAP-1 in lymphoid organs during development
In the primary lymphoid organs very prominent VAP-1 synthesis
was seen in thymus during the fetal life. In particular, the cortical
preponderance of VAP-1 positivity was notable (Figure 2). In bone
marrow, a few vascular sinuses reacted with the anti–VAP-1 mAb,
whereas all other cellular elements lacked this molecule (data
not shown).
In the secondary lymphoid organs, the spleen displayed strikingly bright VAP-1 expression from early on. Of special interest
was to note that the marginal zone sinuses at the margins of white
pulp were VAP-1 positive (Figure 2). It is believed that leukocyte
entry to the spleen takes place mainly via these channels lined by
macrophage-like cells.36 In addition, the dendritic cell–like stromal
component in the splenic follicles synthesized high levels of VAP-1
(Figure 2). VAP-1 was present also in high endothelial venules in
PLNs already in the earliest samples (15 weeks) we managed to
collect (Figure 2).
Nonvascular expression of VAP-1
Figure 1. Time-dependent induction of VAP-1 in parenchymal organs during
fetal development in man. Representative immunohistochemical stainings of
VAP-1 in liver (black arrow indicates central vein; white arrow, sinusoid), heart (black
arrow, capillary), kidney (black arrow, intertubular capillaries; G indicates glomerulus)
and gut (black arrow, gut wall; white arrow, muscularis mucosae; arrowhead,
epithelium) at the indicated weeks of gestation are shown. Negative control (Neg.Co)
stainings from the same specimens are also shown (lower row for each organ). Scale
bar indicates 50 ␮m. Objective magnification for all images, 20⫻.
positivity in these vessels only reached adult levels at birth
(Figure 1). In heart, VAP-1–positive smooth muscle cells were
seen from 11 weeks onwards in the large vessels. Nevertheless,
the small capillaries were initially devoid of VAP-1. During the
second and third trimesters, a very prominent synthesis of
VAP-1 was turned on in these small size vessels (Figure 1).
Kidneys remained VAP-1 negative until week 13. Thereafter,
occasional VAP-1–expressing smooth muscle cells appeared. It
was not before week 23, however, when the peritubular
capillaries started to express VAP-1 (Figure 1). Notably, the
glomerular endothelium remained VAP-1 negative throughout
the life. The lamina propria of intestine was the fourth place
where distinct wave of VAP-1 expression was evident. Thus, in
the maturing gut, the smooth muscle cells (first in the gut wall
from week 7 onwards, and then in large vessels and muscularis
mucosae from week 13 onwards) became VAP-1 positive. In the
villae, in contrast, VAP-1 was absent from the vessels until week
18, although CD31–positive vessels were clearly observed
already at week 9 (Figure 1 and data not shown). Thus, small
caliber vessels in the liver, heart, kidney, and gut, which support
VAP-1–dependent leukocyte recruitment during adult inflamma-
In the developing lung, strong VAP-1 positivity was observed in the
stromal cells in the first samples that we had (13 weeks).
Comparisons with CD31 expression (an endothelial marker) revealed that VAP-1 was prominently present also on nonendothelial
cells (Figure 3). By week 23, the VAP-1 expression became more
restricted to the vessel wall (including the smooth muscle cells) in
the interalveolar septae, but it did not fully colocalize with
CD31–positive endothelial cells, even after birth. In adrenal,
VAP-1 positivity was found in the endocrine cells in the cortex
already at week 13 (our earliest adrenal specimen), and the staining
became more intense at later time points (Figure 3). Notably, the
hormone-synthesizing cells lack VAP-1 in the subcapsular granular
layer, but they acquired VAP-1 expression at the deeper cell layers.
In medulla, again, only the vascular cells showed VAP-1 reactivity.
Figure 2. The expression of VAP-1 in lymphoid organs during the ontogeny in
humans. Representative immunohistochemical stainings of thymus (black arrow
indicates vessel; C, cortex; M, medulla), spleen (black arrow, vessel; arrowhead,
central arteriole; WP, white pulp) and PLN (black arrow, venule; white arrow, high
endothelial venule) are shown. The gestational age (weeks, except the right-hand
panel of a spleen that is 1.5 years) is shown in the top right corner. Scale bar indicates
50 ␮m. Objective magnification for all images, 20⫻.
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SALMI and JALKANEN
Figure 3. Prominent synthesis of VAP-1 in embryonic lung and adrenal gland.
Representative immunohistochemical stainings from fetal (13 weeks) and postnatal
(2 months) lungs and fetal adrenal glands (13 and 23 weeks) are shown. Adjacent
sections of the lungs were stained for VAP-1 and CD31 (an endothelial marker).
Scale bar indicates 50 ␮m. Objective magnification for all images, 20⫻.
VAP-1 dimers are enzymatically active in fetus
In immunoblotting experiments a faint, specific, approximately
180-kDa band was detected with anti–VAP-1 mAb in a lysate made
from a whole 11-week-old fetus. In liver, which mainly contains
the endothelial VAP-1, the electrophoretic mobility of VAP-1 in 17and 40-week-old fetuses was also similar to that seen in adult livers
and tonsils (Figure 4A). The specificity of the signal was further
confirmed by immunodepleting liver lysate with beads armed with
an anti–VAP-1 mAb.
Fetal VAP-1 was able to oxidatively deaminate methylamine, a
model substrate for SSAOs. The level of SSAO activity detectable
in fetal tissues correlated well to the amount of VAP-1 seen in
immunohistochemistry (Figure 4B). Thus, dimerized and enzymatically active VAP-1 is present for at least the 6 last months of the
fetal development in man.
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
Figure 5. Increased levels of soluble, enzymatically active VAP-1 in cord blood.
(A) The levels of soluble VAP-1 protein (sVAP-1; mean ⫾ SEM) in adult and cord
blood were determined using a sandwich ELISA. (B) The SSAO activity of sVAP-1
(mean ⫾ SEM) in sera was determined using the fluorometric assay. (C) The
concentration of sVAP-1 was correlated to the SSAO activity in the 12 cord blood
samples. (D) VAP-1 is the major SSAO enzyme in cord blood. Serum samples were
left untreated or immunodepleted with anti–VAP-1 or control mAb beads, and the
remaining SSAO activity (mean ⫾ SEM; n ⫽ 3) was determined. **P ⬍ .01 between
the control mAb and anti–VAP-1 mAb–depleted samples.
blood than in the blood circulation of adults (Figure 5A). In the
serum collected from the cord blood SSAO enzyme activity was
also readily detected (Figure 5B), and again, it was somewhat
elevated when compared with the adult levels. There was a good
correlation between the sVAP-1 levels and SSAO activity in the
cord blood (Figure 5C). sVAP-1 accounted for the majority of
SSAO activity, since depletion of sVAP-1 from these samples by
sequential immunoprecipitation with anti–VAP-1 mAbs was accompanied with an 80% reduction in the SSAO activity (Figure 5D).
Hence, enzymatically active sVAP-1, which may exert regulatory
functions in leukocyte adhesion, is present in the blood already
before the birth.
Increased soluble SSAO in fetal blood is derived from VAP-1
The concentrations and activity of soluble VAP-1 (sVAP-1) was
determined from sera collected from the cord blood. ELISA assays
showed that sVAP-1 protein levels were slightly higher in the cord
VAP-1 mediates lymphocyte adhesion in newborns
We first analyzed the lymphocyte binding capacity of VAP-1 using
a frozen-section binding assay, which reflects well the specificity
Figure 4. Enzymatically active VAP-1 dimers are synthesized already at week 11 of human gestation. (A) Tissue
lysates were prepared from the indicated fetal and adult
tissues, and analyzed by immunoblotting with anti–VAP-1
mAbs. In addition, the samples from a 40-week liver were
precleared with anti–VAP-1 (VAP-1 depl)– or control mAb
(contr depl)–armed beads, as indicated. The molecularweight markers are indicated on the left. (B) Tissue lysates
were prepared from the indicated tissues. The specific enzymatic SSAO activity was measured using the fluorometric
assay, and is expressed as picomols of H2O2 produced per
1 ␮g protein in 1 hour. The results from a representative
assay, out of 2 with similar results, are shown.
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BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
VAP-1 DURING HUMAN ONTOGENY
1559
in nonlymphoid tissues under nonstatic conditions already
before birth.
Cord blood lymphocytes adhere to VAP-1 in an
enzyme-dependent manner under physiologically relevant
laminar shear stress
Figure 6. Fetal VAP-1 supports lymphocyte binding to venules. The binding of
adult PBLs (A) and cord blood lymphocytes (B-C) to fetal and adult gut and PLN
sections in the presence of anti–VAP-1 or control mAb was determined using the
frozen-section binding assay. The results are mean ⫾ SEM of 3 independent assays.
(D) Adhesion (mean ⫾ SEM) of adult PBLs and cord blood lymphocytes (both from 2
donors) to vessels in 2 different 40-week livers was determined as in panels A-C.
*P ⬍ .05; **P ⬍ .01.
and molecular mechanisms of leukocyte-endothelial interactions
in vivo.37 Guts from 18-week-old fetuses and guts and PLNs from
newborns, in which the expression and function of vascular
addressins PNAd and MadCAM-1 is still of fetal type, were used.38
Adult PBLs adhered well to the venules in these tissues. Both in the
gut and in PLNs, anti–VAP-1 mAbs abrogated about 50% of adult
lymphocyte adhesion to high endothelial venules (HEVs) when
compared with a non–function-blocking control mAb (Figure 6A).
Thus, VAP-1 is functionally intact as an adhesion molecule already
in the fetus.
To see whether lymphocytes in newborns are able to bind
to VAP-1, we isolated cord blood lymphocytes. We first tested
their binding to mature adult VAP-1. These cells bound well to
PLNs and gut HEVs, and in both cases the lymphocyte–
endothelial cell interaction was strongly dependent on VAP-1
(Figure 6B).
The binding of cord blood lymphocytes to fetal-type vessels
was finally tested. Also in this case, anti–VAP-1 treatment inhibited
lymphocyte binding to gut vasculature by about 40%. Even more
profound VAP-1 dependence of cord blood lymphocyte binding to
HEVs was evident in PLNs (Figure 6C).
At 40 weeks, sinusoidal endothelial cells in the liver expressed
high levels of VAP-1 (Figure 1), but enzymatically, it appeared
relatively inactive (Figure 4B). Therefore, we tested whether
VAP-1 is functionally competent in supporting leukocyte adhesion
in these vessels. Using the nonstatic binding assay we saw good
binding of both adult PBLs and cord blood lymphocytes to vessels
of the liver at week 40. An anti–VAP-1 antibody blocked about
40% of binding in both cases (Figure 6D). In similar assays using
the same antibody, a 90% reduction was seen when adult PBLs
binding to adult liver was measured,33 suggesting that either the
expression or adhesive function of VAP-1 further increases later
during the postnatal life concurrently with the appearance of the
full enzymatic activity. Together, these data show that VAP-1 can
mediate lymphocyte binding to endothelial cells in lymphoid and
To mimic the physiologic shear in blood vessels more accurately,
we finally used in vitro capillary flow assay for the binding
experiments. This assay also allows the testing of the contribution
of the enzyme activity of VAP-1 to the adhesion. In this set-up,
HUVECs were adenovirally transfected to express VAP-1 on this
fetal type of endothelial cell. Thereafter, the endothelial cells were
pretreated with anti–VAP-1 mAbs, enzyme inhibitors, or relevant
controls, and the lymphocytes were perfused over the endothelial
cells at a laminar shear of 1.0 dyn/cm.2
When the PBL–VAP-1 interactions were studied it was evident
that cord blood cells interacted with VAP-1–expressing endothelium even better than adult PBLs. On average, cord blood
lymphocytes rolled 1.8 times better, firmly adhered 3.3 times better,
and transmigrated 2.1 times better than adult PBLs (n ⫽ 5-7). As
shown in Figure 7A, the number of rolling cord blood cells was
statistically significantly reduced (by more than 30%) when the
endothelium was pretreated with anti–VAP-1 mAb. The firm
adhesion of cord blood cells was also constantly and significantly
inhibited when blocking the function of VAP-1 with the specific
antibody (Figure 7B-C). It should be noted that a mAb against HLA
class I, which stains the cells brightly without interfering with
leukocyte adhesion, was used as a control. However, anti–VAP-1
mAb did not markedly alter the transmigration of cord blood
lymphocytes through the HUVEC monolayer (Figure 7D). Thus,
Figure 7. VAP-1 supports rolling and firm adhesion of cord blood lymphocytes
under physiologically relevant shear. VAP-1 was transfected into HUVECs, and
the binding of cord blood lymphocytes was determined using the flow chamber assay
under laminar shear stress of 1 dyn/cm.2 HUVECs were activated with TNF-␣, and
the numbers of the rolling cells (A), firmly adherent cells (B-C) (both from the
rolling/firm adhesion protocol [5-minute perfusion] and from the transmigration
protocol [30-minute perfusion]) and transmigrated cells (D) was determined in the
presence of an anti–VAP-1 mAb (TK-8-14), an SSAO inhibitor (5 ␮M hydroxylamine),
a binding but noninhibiting control mAb (against HLA class I), or vehicle, as
appropriate. The results are mean ⫾ SEM of 5 independent assays using different
HUVECs and lymphocytes. *P ⬍ .05; **P ⬍ .01 (all comparisons between the
negative control and the indicated treatment).
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1560
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anti–VAP-1 mAb can diminish cord blood lymphocyte rolling and
adhesion to VAP-1–expressing endothelial cells under physiologically relevant shear.
The prototype SSAO inhibitor hydroxylamine was used to block
SSAO activity of VAP-1–expressing HUVECs. At the concentration
used, it inhibits SSAO activity to the levels seen in cells transfected with
a catalytically inactive VAP-1.8 SSAO inhibition caused a 25% reduction in the number of rolling cells (Figure 7). Interestingly, the SSAO
inhibitor treatment had no effect on the firm adhesion or transmigration
of cord blood lymphocytes. Together, these data suggest that enzyme
activity–dependent and enzyme activity–independent functions of VAP-1
are involved in rolling, whereas only the enzyme activity–independent
functions contribute to the firm adhesion, and that VAP-1 does not seem
to mediate transmigration of cord blood lymphocytes.
Discussion
We report here the first analysis of expression and function of
human VAP-1 during development. The ontogenic regulation of
this molecule has not been studied in any animal model either, and
in fact, it also represents the first developmental characterization of
any member of the SSAO enzyme family. The results show that
VAP-1 synthesis occurs from embryonic week 7 onward in many
tissues and organs. VAP-1 serves its dualistic function already
before birth: it secures lymphocyte binding to venules and it is
catalytically active as an SSAO enzyme. Moreover, the enzymatic
activity of VAP-1 is important in rolling of cord blood lymphocytes
on VAP-1–expressing HUVECs under conditions of physiologically relevant shear conditions.
The mechanisms directing lymphocyte recirculation during the
development have remained mysterious. We have reported earlier
that the mucosal addressin MAdCAM-1 is induced in lymphatic
(and nonlymphatic) tissues at time points (7 weeks) comparable
with VAP-1 induction, whereas the peripheral addressin PNAd
only appears later in week 15.38 MAdCAM-1 and PNAd are
aberrantly expressed in a non–tissue-selective manner during the
fetal development, and they are functionally active at least around
birth.38,39 Hence, many of the vascular adhesion molecules seem to
be induced quite early during the lymphoid development. Hence, it
is possible that these molecules guide initial seeding of lymphoid
cells into the lymphoid tissue anlage. VAP-1 may also contribute to
these homing events. This is suggested by its expression in the
thymic cortex and in the spleen in those vessels used by the
lymphocytes to colonize these organs.
Our data show experimental evidence that fetal type VAP-1 molecule is functionally intact in terms of adhesive and enzymatic activities.
VAP-1 expressed in fetal and neonatal lymphoid organs and on
HUVECs mediated cord blood lymphocyte adhesion both in nonstatic
frozen-section binding assays and in laminar flow assays. This binding
involved the oxidase activity of this cell-surface enzyme inasmuch as
SSAO inhibitors also blocked lymphocyte-endothelial interaction in the
HUVEC model, in which SSAO activity originates solely from the
recombinant VAP-1 molecule. Although due to technical limitations
with human tissues we were not able to directly analyze leukocyteendothelial interactions during earlier development, these data give
support for the concept that endothelial VAP-1 is involved in leukocyte
trafficking during fetal life.
The biological role of SSAO activity has remained enigmatic.
We and others have ascribed a direct role for it in leukocyte rolling,
adhesion, and transmigration.6,7,9-13,40 This finding and molecular
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
modeling of VAP-1 (based on the crystal structure41) implicated
leukocyte cell-surface amines as substrates for SSAO.6 In addition
to cell adhesion, SSAO has been suggested to scavenge exogenous
amines ingested by the alimentary tract.42 Based on the intestinal
induction of VAP-1 in the fetus during pregnancy, VAP-1 becomes
optimally positioned in intestinal vessels already before birth,
although the physiologic consequences of this possible function
remain to be shown. The prominent synthesis of VAP-1 specifically
in smooth muscle cells, but not in other muscle cell types, could
imply that the activity may be associated with maturation of this
particular cell type. VAP-1 is also strongly induced on adipocytes
during early fetal development. In vitro, the expression of VAP-1 is
induced during in vitro differentiation of rodent fibroblastoid cell
lines toward mature adipocytes.21-25 In these cells, the SSAO
activity contributes to the glucose uptake via the GLUT4 transporter, inasmuch as inclusion of the sVAP-1 substrate benzylamine
(in combination with low-dose vanadate treatment) mimics insulinstimulated glucose uptake. Role of VAP-1 in adipocyte metabolism
may be related to its synthesis in endocrine cells in adrenal glands,
in which lipid metabolism is important.
There are 4 SSAO enzymes in man and mice, 1 of which
appears to be a pseudogene.14,18,19,43 In fact, the current analyses of
VAP-1 are the first ontogenic analyses of any of these enzymes in
any species. Based on VAP-1 knockout mice studies VAP-1 is the
major, if not the only, SSAO enzyme in many tissues.13 The
VAP-1–null animals with a strain 129 background do not have any
apparent developmental abnormalities. However, these animals
have not been subjected to any structural and functional analyses
during their development in utero. Therefore, it remains possible
that absence of VAP-1 might cause a mild defect in lymphocyte
recirculation under physiologic conditions or an impaired host
response upon inflammatory challenge, also in utero. Moreover, the
expression of VAP-1 differs between mouse and humans. Most
notably, the liver and kidney, which are brightly VAP-1 positive in
humans, are practically devoid of VAP-1 in mice.28 Therefore,
although the available mouse data argue against a central role for
VAP-1 during ontogeny, the possibility (which is impossible to
verify experimentally) still remains that it may play a role during
human ontogeny. Our results show that the expression of VAP-1 is
turned on early and in a time-dependent manner in different organs
during human fetal development. Once it appears, VAP-1 seems to
be thereafter continuously expressed in the vascular wall during all
later stages of development. Finally, our data indicate that VAP-1
confers enzymatic SSAO activity early during development, and
that it also has adhesive and enzymatic roles in endothelial cells
during intrauterine development in man. VAP-1 is currently being
targeted in preclinical and early clinical trials by antibodies and
small-molecule inhibitors.44-46 Our data imply that VAP-1 may play
a role during fetal development in humans, and therefore they
should be kept in mind if anti–VAP-1 therapies are at some point
planned for use in fertile women.
Acknowledgments
We thank Dr Kalle Alanen for providing the tissue material, Kaisa
Koskinen for advice with the flow assay, Ms Pirjo Heinilä and
Riikka Sjöroos for expert technical assistance, and Ms Anne
Sovikoski-Georgieva for secretarial help.
From www.bloodjournal.org by guest on June 15, 2017. For personal use only.
BLOOD, 1 SEPTEMBER 2006 䡠 VOLUME 108, NUMBER 5
VAP-1 DURING HUMAN ONTOGENY
1561
References
1. von Andrian UH, Mempel TR. Homing and cellular traffic in lymph nodes. Nat Rev Immunol.
2003;3:867-878.
ing, and characterization of the promoter. J Biol
Chem. 1994;269:14484-14489.
33. McNab G, Reeves JL, Salmi M, Hubscher S, Jalkanen S, Adams DH. Vascular adhesion protein 1
mediates binding of T cells to human hepatic endothelium. Gastroenterology. 1996;110:522-528.
2. Muller WA. Leukocyte-endothelial-cell interactions in
leukocyte transmigration and the inflammatory response. Trends Immunol. 2003;24:327-334.
19. Imamura Y, Kubota R, Wang Y, et al. Human
retina-specific amine oxidase (RAO): cDNA cloning, tissue expression, and chromosomal mapping. Genomics. 1997;40:277-283.
3. Salmi M, Jalkanen S. A 90-kilodalton endothelial
cell molecule mediating lymphocyte binding in
humans. Science. 1992;257:1407-1409.
20. Jaakkola K, Kaunismäki K, Tohka S, et al. Human
vascular adhesion protein-1 in smooth muscle
cells. Am J Pathol. 1999;155:1953-1965.
34. Jaakkola K, Jalkanen S, Kaunismäki K, et al. Vascular adhesion protein-1, intercellular adhesion
molecule-1 and P- selectin mediate leukocyte
binding to ischemic heart in humans. J Am Coll
Cardiol. 2000;36:122-129.
4. Salmi M, Kalimo K, Jalkanen S. Induction and
function of vascular adhesion protein-1 at sites of
inflammation. J Exp Med. 1993;178:2255-2260.
21. Enrique-Tarancón G, Marti L, Morin N, et al. Role
of semicarbazide-sensitive amine oxidase on glucose transport and GLUT4 recruitment to the cell
surface in adipose cells. J Biol Chem. 1998;273:
8025-8032.
35. Kurkijärvi R, Jalkanen S, Isoniemi H, Salmi M.
Vascular adhesion protein-1 (VAP-1) mediates
lymphocyte-endothelial interactions in chronic
kidney rejection. Eur J Immunol. 2001;31:
2876-2884.
22. Moldes M, Fève B, Pairault J. Molecular cloning
of a major mRNA species in murine 3T3 adipocyte lineage: differentiation-dependent expression, regulation, and identification as semicarbazide-sensitive amine oxidase. J Biol Chem. 1999;
274:9515-9523.
36. Mebius RE, Kraal G. Structure and function of the
spleen. Nat Rev Immunol. 2005;5:606-616.
5. Salmi M, Jalkanen S. Human vascular adhesion
protein-1 (VAP-1) is a unique sialoglycoprotein
that mediates carbohydrate-dependent binding of
lymphocytes to endothelial cells. J Exp Med.
1996;183:569-579.
6. Salmi M, Yegutkin G, Lehvonen R, Koskinen K,
Salminen T, Jalkanen S. A cell surface amine oxidase directly controls lymphocyte migration.
Immunity. 2001;14:265-276.
7. Lalor PF, Edwards S, McNab G, Salmi M, Jalkanen S, Adams DH. Vascular adhesion protein-1
mediates adhesion and transmigration of lymphocytes on human hepatic endothelial cells. J Immunol. 2002;169:983-992.
8. Koskinen K, Vainio PJ, Smith DJ, et al. Granulocyte transmigration through endothelium is regulated by the oxidase activity of vascular adhesion
protein-1 (VAP-1). Blood. 2004;103:3388-3395.
9. Bonder C, Swain MG, Zbytnuik LD, et al. Rules of
recruitment of trafficking Th1 and Th2 cells in inflamed liver. Immunity. 2005;23:153-163.
23. Abella A, Marti L, Camps M, et al. Semicarbazidesensitive amine oxidase/vascular adhesion protein-1
activity exerts an antidiabetic action in Goto-Kakizaki
rats. Diabetes. 2003;52:1004-1013.
24. Morin N, Visentin V, Calise D, et al. Tyramine
stimulates glucose uptake in insulin-sensitive tissues in vitro and in vivo via its oxidation by amine
oxidases. J Pharmacol Exp Ther. 2002;303:
1238-1247.
25. Fontana E, Boucher J, Marti L, et al. Amine oxidase substrates mimic several of the insulin effects on adipocyte differentiation in 3T3 F442A
cells. Biochem J. 2001;356:769-777.
10. Tohka S, Laukkanen M-L, Jalkanen S, Salmi M.
Vascular adhesion protein 1 (VAP-1) functions as
a molecular brake during granulocyte rolling and
mediates their recruitment in vivo. FASEB J.
2001;15:373-382.
26. Kurkijärvi R, Adams DH, Leino R, Möttönen T,
Jalkanen S, Salmi M. Circulating form of human
vascular adhesion protein-1 (VAP-1): increased
serum levels in inflammatory liver diseases. J Immunol. 1998;161:1549-1557.
11. Martelius T, Salmi M, Wu H, et al. Induction of
vascular adhesion protein-1 during liver allograft
rejection and concomitant cytomegalovirus infection in rats. Am J Pathol. 2000;157:1229-1237.
27. Kurkijärvi R, Yegutkin GG, Gunson BK, Jalkanen
S, Salmi M, Adams DH. Circulating soluble vascular adhesion protein 1 accounts for the increased serum monoamine oxidase activity in
chronic liver disease. Gastroenterology. 2000;
119:1096-1103.
12. Merinen M, Irjala H, Salmi M, Jaakkola I, Hanninen A, Jalkanen S. Vascular adhesion protein-1 is
involved in both acute and chronic inflammation
in the mouse. Am J Pathol. 2005;166:793-800.
13. Stolen CM, Marttila-Ichihara F, Koskinen K, et al.
Absence of the endothelial oxidase AOC3 leads
to abnormal leukocyte traffic in vivo. Immunity.
2005;22:105-115.
14. Smith DJ, Salmi M, Bono P, Hellman J, Leu T,
Jalkanen S. Cloning of vascular adhesion protein-1 reveals a novel multifunctional adhesion
molecule. J Exp Med. 1998;188:17-27.
15. Salmi M, Jalkanen S. Cell-surface enzymes in
control of leukocyte trafficking. Nat Rev Immunol.
2005;5:760-771.
16. Klinman JP, Mu D. Quinoenzymes in biology.
Annu Rev Biochem. 1994;63:299-344.
17. Jalkanen S, Salmi M. Cell surface monoamine
oxidases: enzymes in search of a function.
EMBO J. 2001;20:3893-3901.
18. Chassande O, Renard S, Barbry P, Lazdunski M.
The human gene for diamine oxidase, an amiloride binding protein: molecular cloning, sequenc-
28. Bono P, Jalkanen S, Salmi M. Mouse vascular
adhesion protein-1 (VAP-1) is a sialoglycoprotein
with enzymatic activity and is induced in diabetic
insulitis. Am J Pathol. 1999;155:1613-1624.
29. Parham P. Monoclonal antibodies against two
separate alloantigenic sites of HLA-B40.
Immunogenetics. 1981;13:509-527.
30. Streeter PR, Berg EL, Rouse BTN, Bargatze RF,
Butcher EC. A tissue-specific endothelial cell molecule involved in lymphocyte homing. Nature.
1988;331:41-46.
31. Salmi M, Tohka S, Berg EL, Butcher EC, Jalkanen S. Vascular adhesion protein 1 (VAP-1)
mediates lymphocyte subtype-specific, selectinindependent recognition of vascular endothelium
in human lymph nodes. J Exp Med. 1997;186:
589-600.
32. Salmi M, Koskinen K, Henttinen T, Elima K, Jalkanen S. CLEVER-1 mediates lymphocyte transmigration through vascular and lymphatic endothelium. Blood. 2004;104:3849-3857.
37. Butcher EC, Scollay RG, Weissman IL. Lymphocyte
adherence to high endothelial venules: characterization of a modified in vitro assay, and examination of the binding of syngeneic and allogeneic
lymphocyte populations. J Immunol. 1979;123:
1996-2003.
38. Salmi M, Alanen K, Grenman S, Briskin M,
Butcher EC, Jalkanen S. Immune cell trafficking
in uterus and early life is dominated by the mucosal addressin madcam-1 in humans. Gastroenterology. 2001;121:853-864.
39. Mebius RE, Streeter PR, Michie S, Butcher EC,
Weissman IL. A developmental switch in lymphocyte homing receptor and endothelial vascular
addressin expression regulates lymphocyte homing and permits CD4⫹CD3- cells to colonize
lymph nodes. Proc Natl Acad Sci U S A. 1996;93:
11019-11024.
40. Yoong KF, McNab G, Hübscher SG, Adams DH.
Vascular adhesion protein-1 and ICAM-1 support
the adhesion of tumor- infiltrating lymphocytes to
tumor endothelium in human hepatocellular carcinoma. J Immunol. 1998;160:3978-3988.
41. Airenne TT, Nymalm Y, Kidron H, et al. Crystal
structure of the human vascular adhesion protein-1: unique structural features with functional
implications. Protein Sci. 2005;14:1964-1974.
42. Yu PH, Wright S, Fan EH, Lun ZR, GubisneHarberle D. Physiological and pathological implications of semicarbazide-sensitive amine oxidase. Biochim Biophys Acta. 2003;1647:193-199.
43. Cronin CN, Zhang X, Thompson DA, McIntire
WS. cDNA cloning of two splice variants of a human copper-containing monoamine oxidase
pseudogene containing a dimeric Alu repeat sequence. Gene. 1998;220:71-76.
44. Vainio PJ, Kortekangas-Savolainen O, Mikkola
JH, et al. Safety of blocking vascular adhesion
protein-1 in patients with contact dermatitis. Basic
Clin Pharmacol Toxicol. 2005;96:429-435.
45. Salter-Cid LM, Wang E, O’Rourke AM, et al. Antiinflammatory effects of inhibiting the amine oxidase activity of semicarbazide-sensitive amine
oxidase. J Pharmacol Exp Ther. 2005;315:
553-562.
46. Kirton CM, Laukkanen ML, Nieminen A, et al.
Function-blocking antibodies to human vascular
adhesion protein-1: a potential anti-inflammatory
therapy. Eur J Immunol. 2005;35:3119-3130.
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2006 108: 1555-1561
doi:10.1182/blood-2005-11-4599 originally published online
March 23, 2006
Developmental regulation of the adhesive and enzymatic activity of
vascular adhesion protein-1 (VAP-1) in humans
Marko Salmi and Sirpa Jalkanen
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