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Regular Articles |
From the National Public Health Institute and MediCity Research
Laboratory,*
University of Turku, Turku; the Departments of
Medicine
and
Surgery,
Turku University Central Hospital,
Turku; the Laboratory of Electron Microscopy,§
University of Turku, Turku; and the Department of
Pathology,¶
Helsinki University Central Hospital,
Helsinki, Finland
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition to endothelial cells, VAP-1 is also present in smooth muscle cells.1,2 However, there is no detailed knowledge of the expression pattern of VAP-1 in different types of muscle cells, and the regulation of VAP-1 in pathological states involving smooth muscle cells, like tumorigenesis and atherogenesis, has not been assessed. Moreover, the structural similarity between VAP-1 in endothelial cells and VAP-1 in muscle cells has not been studied, and the function of VAP-1 at nonendothelial sites has remained elusive. There is at least one other endothelial adhesion molecule, vascular cell adhesion molecule-1 (VCAM-1, CD106), which is also expressed on these two distinct cell types.6-8 Interestingly, expression of VCAM-1 in endothelial cells and muscle cells is differentially regulated,9,10 and VCAM-1 serves different functions in these two cell types.6,8 To understand the role of VAP-1 in muscle cells, we undertook a study to evaluate the localization, structure, and function of VAP-1 in different types of muscle cells.
We report here that VAP-1 is selectively expressed in smooth muscle among different types of muscle cells. In these cells, it is present on specialized plasma membrane domains as dimeric and trimeric forms, and it is a heavily glycosylated protein. VAP-1 expression is modulated during malignant transformation of smooth muscle cells. Functionally, smooth muscle VAP-1 displays clear differences from endothelial VAP-1 in serving only an enzymatic but not an adhesive function. Together, these data imply that VAP-1 molecules in different cell types are structurally distinct and serve different functions.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Muscle samples (the major saphenous vein, aorta, heart, gut from small intestine, appendix and colon, and bladder wall) were obtained from surgical operations, and skeletal muscle and large vessels (vena cava) were from autopsies. All of these tissues were macroscopically and microscopically normal. Tonsil samples were from tonsillectomies. Aortic smooth muscle cells (CRL 1999) and smooth muscle cell lines HISM (human intestinal smooth muscle, CRL 1692) and SK-LMS-1 (leiomyosarcoma, HTB 88) were from the American Type Culture Collection and cultured in RPMI 1640 containing 10% fetal calf serum (Gibco BRL, Paisley, UK), 2 mmol/L L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Peripheral blood lymphocytes (PBLs) were isolated from normal volunteers, using Ficoll-Hypaque gradient centrifugations.
Tumors, Atheromatous Samples, and Inflamed Tissue Specimens
Benign leiomyomas were from uterine tissue, and malignant
leiomyosarcomas were collected from different locations as detailed in
Table 1
. A portion of the specimens was
snap-frozen in liquid nitrogen, and the rest was submitted to routine
histopathological analyses. The samples from atheromatous arteries were
taken during five surgical correction procedures of atheromatous aortic
aneurysms. The specimens were taken from the borderline between
aneurysmal and normal areas of arteries. The control specimens
representing normal arterial wall were from macroscopically and
microscopically nonatheromatous arteries of five patients (four renal
arteries and one splenic artery) who underwent surgery for nonvascular
indications. In addition, surgical samples from severely inflamed gut
with massive leukocyte infiltrations (ulcerative colitis) were obtained
from colectomy operations.
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A function-inhibiting mouse mAb 1B2 against human VAP-1 and a control mAb against chicken T cells have been described.1 A new mAb 2D10 (mouse IgG1) was raised against immunoaffinity-purified VAP-1.11 Because neither 1B2 nor 2D10 works in formalin-fixed, paraffin-embedded sections, use of frozen sections was mandatory in all experiments.
Function blocking mAbs TS1/18 against ß2-integrins
(CD18),12
HP2/1 against 
4-integrin
(CD49d),13
and Hermes-1 and Hermes-3 against the
hyaluronan-binding and endothelial-binding epitopes of
CD4414
have been described. To identify endothelial cells
mAb 2C8 against CD3115
and a polyclonal rabbit anti-human
Factor VIII (Dakopatts, Glostrup, Denmark) were used.
Peroxidase-conjugated sheep anti-mouse Ig was from Dakopatts, and
fluorescein isothiocyanate-conjugated sheep anti-mouse Ig was from
Sigma.
Immunolabeling and Electron Microscopy
Immunocytochemical reactions on acetone-fixed frozen sections were made by using an indirect immunoperoxidase method for light microscopy as described earlier.1 After detachment of adherent cells with 5 mmol/L EDTA, analyses of smooth muscle cells in suspension were done with and without permeabilization (-20°C acetone, 2 minutes) by indirect immunofluorescence and fluorescence-activated cell sorter, using an earlier described protocol.16 Reactions of adherent cells were carried out on chamber slides as described.16
For immunolabeling electron microscopy, 1–2-mm-thick slices were cut from the tissue samples under a stereomicroscope. Selected regions were fixed for 3 hours in 2% paraformaldehyde fixative buffered with Sörensen’s phosphate buffer (pH 7.4). Then the specimens were incubated in 2.1 mol/L sucrose in water for 48 hours, embedded in O.C.T. (Tissue-Tec; Miles, Elkhart, IN), and frozen in liquid nitrogen. Sixty-micron frozen sections were cut, and the immunocytochemical reactions were made in free floating sections in test tubes. Anti-VAP-1 mAbs 1B2 and 2D10 were used at 5–10 µg/ml, and 2% normal mouse serum was used as a negative control. Incubation time was 48 hours for primary antibodies and 24 hours for the peroxidase-conjugated secondary antibody at 4°C. The washes between the antibody incubations were 3 x 45 minutes. The reaction was visualized using 3,3'-diamino-benzidine (DAB)-tetrahydrochloride (Polysciences, Warrington, PA) as an electron donor. Although the DAB precipitate becomes electron opaque in the osmium fixative, in several specimens the immunoreaction was further intensified with silver enhancement, as described.17,18 In brief, precipitated DAB was first treated for 5 minutes with 0.1% sodium sulfide and washed in 250 mmol/L acetate buffer (pH 7.0). Then the reaction was developed in a solution containing sodium carbonate, ammonium nitrate, silver nitrate, tungstosilicic acid, and formaldehyde. The reaction was stopped with 1% acetic acid, and the product was stabilized by a 3-minute treatment with 0.05% gold chloride. After a 5-minute incubation in 0.3% sodium thiosulfate, the specimens were washed in phosphate-buffered saline (PBS) and fixed in 5% glutaraldehyde in 0.16 mol/L s-collidin-HCl buffer (pH 7.4) overnight. Selected specimens containing blood vessels were then postfixed for 2 hours in 1% aqueous OsO4 and processed for embedding in epoxy resin (Glycidether 100; Merck AG, Darmstadt, Germany) as described.19 Thin sections were cut with a Reichert Ultracut E (Reichert-Jung Optische Werke, Vienna, Austria) ultramicrotome, and the grids were examined with a Jeol JEM 100 SX transmission electron microscope.
Immunoblotting and Glycosidase Digestions
Blood vessels were opened with scissors, and they were denuded
from the endothelial layer by mechanical scraping. Lamina propria cells
were stripped from gut samples by mechanical dissection and scraping.
Thereafter, samples from vessel and gut wall, tonsil stroma, heart, and
skeletal muscle were minced and lysed in a buffer containing 1% NP-40,
1.5 mmol/L MgCl2, 10 mmol/L Tris-base (pH 7.4), and 150
mmol/L NaCl or in PBS containing 1% NP-40. After clarification by
centrifugation, the supernatants were treated with exo- and
endoglycosidases or mock treated using an earlier described
protocol.3
Vibrio cholerae sialidase is active
against 2,3, 2,6, and 2,8 linked sialic acids,20
whereas Newcastle disease virus neuraminidase only hydrolyzes 2,3
(and 2,8)-linked sialic acids.21
O-Sialoglycoprotease endopeptidase degrades mucin-like
glycoproteins containing adjacent O-linked sialylated side
chains.22,23
O-Glycans were removed from
sialidase-treated samples with recombinant O-glycanase and
for N-glycanase treatment peptide: N-glycosidase
F was added to PBS-lysate. The specificity and effectiveness of these
digestions were documented earlier.3
Metabolic Labelings
Small fragments of smooth muscle from intestinal wall were labeled with [35S]methionine/cysteine, lysed, and used for immunoprecipitations as described earlier for labeling of lymphoid tissues.3 After washings, the antigens were eluted in Laemmli’s reducing sample buffer and separated by 5–12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The gels were finally fixed, soaked in Enlightning, dried, and autoradiographed.
Adhesion Assays
In a modification of the Stamper-Woodruff endothelial cell binding assay,24 frozen sections of appendices from normal and inflamed intestine and tonsil were cut and overlaid with saturating concentrations of anti-VAP-1 mAb 1B2 or a control mAb. Freshly isolated PBLs without or with phorbol myristate acetate activation (50 ng/ml for 30 minutes at 37°C followed by two washes) were resuspended in RPMI 1640 containing 10% fetal calf serum and 10 mmol/L HEPES (= binding medium). After filtering through a silk mesh, 3 x 106 cells were applied to the sections and allowed to bind for 30 minutes at +7°C under rotation (60 rpm on an orbital shaker) or at 37°C under stationary conditions. The nonadherent cells were then tilted off, and the adherent ones were fixed to the sections with ice-cold 1% glutaraldehyde in PBS. The number of cells bound to HEVs and to smooth muscle cells was counted under dark-field microscopy. Although the VAP-1 dependence of PBL adhesion to endothelial cells is much more pronounced in tonsil than in appendix,1 the use of appendix allowed us to analyze simultaneously the binding of PBLs to HEVs and to muscular wall under identical experimental conditions.
Alternatively, vascular explants (~2–3 cm-long fragments of
saphenous vein) were cut open, and the endothelial cell layer was
stripped off with gentle scraping. The explants were attached from
their corners to the bottom of soft wells by fine needles and
preincubated with an anti-VAP-1 mAb 1B2 or with a negative control mAb
3G6 (100 µg/ml) for 30 minutes. After washing with PBS, 4 x
106
lymphocytes were added into each well in 2 ml of the
binding medium and allowed to adhere to smooth muscle cells under
static or rotatory conditions for 30 minutes at room temperature. The
nonadherent cells were removed by gently inverting the assembly, and
the bound cells were fixed to the tissue by an overnight incubation in
5% glutaraldehyde in PBS. After Diff-Quick staining the number of
adherent lymphocytes was counted from at least seven random microscopic
fields per explant (magnification: x200; 361 ± 89 (mean ±
SEM) bound cells in the negative control mAb-treated explants in four
independent assays). As a positive control, PBLs were preincubated with
mAbs against CD44, CD18, and 4-integrins (each mAb at 25 µg/ml
concentration, 0.5 ml for 10 x 106
cells), washed,
and resuspended in the assay medium, and the adhesion assay was
performed as described above.
Enzymatic Analyses
Amine oxidase activity in smooth muscle was determined radiochemically based on the method of Fowler and Tipton,25 using labeled benzylamine ([7-14C]benzylamine hydrochloride, sp. act. 57 mCi/mmol; Amersham) as a substrate. The standard assay was performed at 37°C for 60 minutes in a final volume of 500 µl of 0.1 mol/L Na-phosphate buffer (pH 7.4) containing smooth muscle lysate (60–100 µg protein); various concentrations of benzylamine with tracer [14C]benzylamine (40,000 dpm); and 1 mmol/L unlabeled tyramine, tryptamine, histamine, methylamine, or ß-phenylethylamine as a competitor. In the inhibitory studies, the lysate was preincubated with 1 mmol/L clorgyline or semicarbazide, preferential inhibitors of SSAO and MAOA/B activity, respectively,5 at 37°C for 30 minutes before benzylamine was added. In certain experiments, smooth muscle lysate was depleted of VAP-1 by immunochromatography. In brief, aliquots of lysate were incubated three times with CnBr-activated Sepharose 4CL beads to which either an anti-VAP-1 or a control antibody had previously been coupled. A complete and specific depletion of VAP-1 by anti-VAP-1 but not by control beads was confirmed by immunoblotting. In all enzyme assays the catalytic reaction was stopped by 120 µl of 2 mol/L citric acid, and aldehyde reaction products were extracted from the analyzed mixture into toluene containing 0.35 g/L diphenyloxazole. The amount of 14C-labeled benzaldehyde was quantified by scintillation counting with a Wallac-1409 ß-counter (Turku, Finland). The activity of the enzyme was expressed as nanomoles of benzaldehyde oxidized by 1 mg lysate protein per hour. Protein concentrations were determined with a bicinchoninic acid assay (Pierce). The kinetic parameters for smooth muscle monoamine oxidase (Km and Vmax) were calculated using a nonlinear curve-fitting program based on the Michaelis-Menten equation (Enzkin, GraphPad Prism).
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Results |
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Tissue distribution of VAP-1 was determined using two different
anti-VAP-1 mAbs, 1B2 and 2D10. The two mAbs gave similar results, but
mAb 2D10 displayed higher intensity. Immunohistochemistry showed that
VAP-1 is absent from skeletal and cardiac muscle cells. In these
tissues only endothelial cells of small vessels and medial cells of the
small arteries and veins were VAP-1 positive (Figure 1, A and C)
. In larger veins VAP-1 was
abundantly present in smooth muscle cells of the medial layer (Figure 1E)
. In the largest vessels (aorta and veins draining into heart), the
endothelium and intimal layer were VAP-1 negative, and this molecule
was absent from the outermost adventitial layer as well. In contrast,
the smooth muscle cells of the medial layer were VAP-1 positive (Figure 1G)
. In other sources of smooth muscle (small intestine, appendix
(Figure 1I)
, skin, bladder wall, uterus) VAP-1 was expressed in a
similar pattern on all smooth muscle cells. VAP-1 reactivity appeared
to surround the surface of the myocyte, being most evident in the cross
sections of smooth muscle fascicles (Figure 1I)
. The reaction pattern
would be compatible with the VAP-1 expression on the plasma membrane of
the myocyte, in the basement membrane, which surrounds each smooth
muscle cell, or in the adjacent extracellular components of the smooth
muscle fibers.
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. It was also present in small
cup-like invaginations of the membrane called caveolae, which,
characteristically of micropinocytotic vesicles, lack any evident
cytoplasmic coat (Figure 2B)
. Thus VAP-1 is expressed in smooth muscle
cells, and it is present on the surface of the cells.
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In endothelial cells VAP-1 is an inflammation-inducible adhesion molecule in vivo.2 To see whether VAP-1 is similarly regulated in smooth muscle cells, we studied atheromatous samples. In these specimens equal VAP-1 positivity was seen in apparently normal smooth muscle cells surrounding the lesion and in those of the atheromatous areas. Moreover, the intensity of VAP-1 in smooth muscle cells of the lesion was comparable to those in the noninvolved segment of the same vessels. The endothelial lining covering the normal and atheromatous large arteries was VAP-1 negative. In another model of inflammation involving smooth muscle cells, specimens from inflammatory bowel disease showed no induction of VAP-1 on gut wall muscle cells, although it was up-regulated in endothelial cells of lamina propria. Thus smooth muscle VAP-1, in contrast to endothelial VAP-1, is not induced on inflammation.
Smooth Muscle Cells Synthesize VAP-1 Only in Vivo
VAP-1 was also searched for in different cell lines of smooth muscle origin. However, both the cell surface and the cytoplasm of intestinal and aortic smooth muscle cells and a leiomyosarcoma line were constantly negative for VAP-1, as detected by immunofluorescence with and without permeabilization. Moreover, smooth muscle cell monolayers derived from tissue explants or by enzymatic dispersion did not display any VAP-1 (data not shown).
Smooth Muscle VAP-1 Is Down-Regulated during Malignant Transformation
Because smooth muscle cell lines derived from tumors were VAP-1
negative, we analyzed the expression of this molecule in
situ in tumor specimens (Table 1
and Figure 3
). In leiomyomas, the expression of
VAP-1 was retained at normal levels. In contrast, two different
expression patterns were observed in leiomyosarcoma samples. In four of
10 specimens, the VAP-1 was clearly present on malignant cells. In six
of 10 samples, however, VAP-1 was completely lost from the
leiomyosarcoma cells, although it was still present on smooth muscle
cells of the vessels, which served as a useful internal standard of the
staining intensity. Thus, at a certain stage of malignant
transformation, smooth muscle cells can stop synthesis of VAP-1.
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In endothelium, VAP-1 is a dimeric protein consisting of two
identical 90-kd subunits.3
To see whether VAP-1 in smooth
muscle cells is biochemically similar, small pieces of smooth muscle
from the gut were labeled in vitro with
[35S]methionine/[35S]cysteine.
Immunoprecipitations with mAb 1B2 showed specific bands at ~90 and
~170 kd under reducing conditions (Figure 4)
. The low-molecular-mass bands
precipitated with 1B2 but not with a control mAb probably are not
specific, inasmuch as they were not seen with anti-VAP-1 mAb 2D10. In
contrast, faint but specific signals detected with both anti-VAP-1 mAbs
were also apparent at higher molecular masses.
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The dimeric form migrates as a 160–185-kd band and the trimeric form
as a 240–260-kd band; hereafter these will be called the 165-kd and
250-kd forms of VAP-1, respectively, according to the estimated size of
the most intense signal. The majority of VAP-1 in skeletal muscle,
heart, and tonsil is of endothelial origin, and it has a slightly
slower electrophoretic mobility than that of smooth muscle preparations
of vessels and gut (Figure 5)
. It should be noted that although the
size differences in immunoblots appear quite small, all samples have
been analyzed in parallel lanes in one gel, and the results were
constant in several independent analyses. Moreover, a small shift in
the electrophorectic mobility at the high-molecular-mass range in
SDS-PAGE already translates into a marked difference in the actual size
of the molecule. Neither mAb 1B2 nor 2D10 reacts with the 90-kd monomer
of VAP-1 in immunoblotting.3
Thus VAP-1 in smooth muscle
cells appears to be mainly in a dimeric form like that in tonsil HEVs.
However, the size difference of smooth muscle VAP-1 and endothelial
VAP-1 suggests that either the protein core is different or there are
alterations in the posttranslational modifications of VAP-1 between the
two cell types.
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Smooth muscle VAP-1 was digested with different exo- and
endoglycosidases to study the extent of posttranslational modifications
in VAP-1 from this source. After V. cholerae sialidase
treatment, which cleaves sialic acids in all common linkages, VAP-1
displayed retarded electrophoretic mobility (Figure 6)
. This
paradoxical decrease in migration in SDS-PAGE after sialidase treatment
is characteristic of abundantly sialylated proteins, and it is
attributed to the removal of large quantities of negatively charged
sialic acids from the molecule. Specific removal of 2,3 (and
2,8)-linked sialic acids with Newcastle disease virus sialidase also
increased the apparent molecular mass of VAP-1, but to a lesser extent.
Both the 165-kd dimeric and the higher molecular mass multimer showed
altered migration in SDS-PAGE after removal of sialic acids (Figure 6)
.
O-Sialoglycoprotein endopeptidase digestion also increased
the molecular mass of both VAP-1 species (Figure 6)
.
Smooth muscle VAP-1 has no major O-linked side chains, inasmuch as
further digestion of sialidase-treated VAP-1 with
O-glycanase had only a minor additional effect
(decrease) on the mobility of VAP-1 when compared to the sialidase
treatment only (Figure 6)
. In contrast, cleavage of N-linked sugars
with recombinant N-glycanase markedly decreased (about 15
kd) the apparent molecular mass of smooth muscle VAP-1 (Figure 6
; the
arrow and arrowhead indicate the mobilities of untreated and
N-glycanase-treated smooth muscle VAP-1, respectively).
Together these data indicate that VAP-1 has abundant sialic acid
decorations and more than one N-linked oligosaccharide side chain.
The effect of these enzymatic treatments was similar in smooth muscle
lysates from all types of origins tested, ie, muscular wall of the
small intestine and wall of medium-sized vein (Figure 7
and data not
shown). These data suggest that smooth muscle VAP-1 in different
tissues and organs is similarly modified. However, it displays several
differences from tonsil VAP-1. The behavior of tonsil VAP-1 is shown as
an internal control in Figures 5–7
to allow direct comparison of VAP-1
of smooth muscle and HEV origin in the same experiments and in the same
SDS-PAGE gels. Notably, tonsillar VAP-1 constantly had a slower
electrophoretic mobility, the trimeric VAP-1 was never seen in tonsil,
and, under these same conditions, tonsil VAP-1 was not sensitive to
N-glycanase.
Smooth Muscle VAP-1 Does Not Bind Lymphocytes
Because endothelial VAP-1 is a sialic acid-dependent adhesion
molecule for blood-borne lymphocytes,3
we determined
whether smooth muscle VAP-1, which appeared to carry the critical
proadhesive modifications, would also support lymphocyte binding. When
PBLs were preincubated on frozen sections under conditions that
resulted in a VAP-1-dependent adherence to vessel endothelium in both
tonsil and appendix, we saw no specific adhesion to smooth muscle cells
in sections of either vascular or gut origin (Figure 8)
. Because these data may reflect
the physiological absence of normal PBLs from these locations, we
preactivated PBLs with phorbol myristate acetate to simulate conditions
of inflammation where lymphocytes can be found in vessel or gut wall.
However, not even under these conditions, at either 7°C or 37°C,
was any significant binding of PBLs to smooth muscle detectable.
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. Specific
interactions of single lymphocytes with the smooth muscle substratum
were readily discernible (Figure 8B)
in the presence of a control mAb.
However, no VAP-1-inhibitable adhesion was seen in this model either
(Figure 8C)
. As a positive control, lymphocytes were preincubated with
a pool of mAbs against CD44, LFA-1, and 4-integrins, adhesion
molecules known to be involved in PBL-smooth muscle cell
interaction.26
A 50% reduction of PBL binding to smooth
muscle cells with these antibodies was observed. These functional data
indicate that smooth muscle VAP-1 may not play a role in PBL adhesion. Smooth Muscle VAP-1 Is a Monoamine Oxidase
Cloning and sequencing of VAP-1 revealed that it belongs to the
SSAO family.4
Therefore, we investigated whether the
smooth muscle expressing high levels of VAP-1 possesses monoamine
oxidase activity and whether it is derived from VAP-1. Lysates made of
gut smooth muscle cells exhibited a significant monoamine oxidase
activity, which displays Michaelis-Menten kinetics, with a
Km for benzylamine of 74 µmol/L (Figure 9A)
. Since benzylamine can be metabolized
by both SSAO and MAO (especially MAO-B), the lysates were pretreated
with selective inhibitors of these enzymes.5
As shown in
Figure 9B
, smooth muscle monoamine oxidase activity was practically
abolished by specific SSAO inhibitor semicarbazide, whereas about 90%
of the enzyme remained insensitive to clorgyline, a MAO inhibitor.
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To study the contribution of VAP-1 to the overall SSAO activity in the
smooth muscle cells, VAP-1 was depleted from the lysate by
immunoaffinity chromatography. As shown in the inset of Figure 9B
, 89%
of SSAO activity was concomitantly abolished, indicating that VAP-1 is
the major but not the only SSAO species in human smooth muscle cells.
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VAP-1 was found in smooth muscle cells in all tissues and organs tested. In contrast, all samples from skeletal muscle and heart only displayed VAP-1 reactivity in blood vessels, whereas striated and cardiac muscle cells were absolutely VAP-1 negative. Therefore, VAP-1 appears to be a useful marker of normal cells of smooth muscle origin. In smooth muscle tumors, VAP-1 was present in benign lesions. In malignant cases VAP-1 expression was either retained or lost. Disappearance of VAP-1 did not appear to correlate with the anatomical location or the differentiation level of the tumor, although the small number of samples of these rare malignancies prevented any statistical analyses. It will be interesting to see in larger series whether VAP-1 positivity can serve as an independent factor in the prognosis of leiomyosarcomas. In smooth muscle cells VAP-1 was clearly localized on the cell surface. Moreover, it was seen in caveolae, bottle-shaped invaginations of the plasma membrane. Caveolae are specialized microdomains of plasma membrane that may have a role in transcytosis of macromolecules, in potocytosis, and in signal transduction.27 Caveolar localization of VAP concentrates this molecule in a discrete region of the plasma membrane.
There are distinct structural differences in VAP of endothelial and
smooth muscle origin. First, smooth muscle VAP-1 displayed faster
apparent electrophoretic mobility than endothelial VAP-1. Second, only
the monomeric and dimeric forms of VAP-1 are found in endothelial
cells.3
In addition to these two forms, trimeric VAP-1
species were detected in smooth muscle. These species of VAP-1
apparently represent multimeric complexes of VAP-1 that are only
partially sensitive to the reduction of disulfide bonds with 5%
2-mercaptoethanol. Third, VAP-1 in smooth muscle cells contained
readily cleavable N-linked glycans, which were absent from the VAP-1 of
endothelial cell origin under the same conditions. Both endothelial and
smooth muscle VAP-1 are abundantly sialylated glycoproteins that result
in aberrant migration in SDS-PAGE gels. Therefore, the reduction of
VAP-1 molecular mass by about 15 kd after N-glycanase
treatment does not allow direct estimation of the number of N-linked
glycans in smooth muscle cells, because retarded mobility caused by
removal of N-linked oligosaccharides may be counteracted by a
paradoxical increase in mobility by simultaneous deletion of sialic
acids on these glycans. Smooth muscle VAP-1 has both 2,3- and
2,6-linked sialic acids, inasmuch as the apparent shift in molecular
mass was less with an 2,3-linkage-specific Newcastle disease virus
neuraminidase than with V. cholerae neuraminidase, which has
a broad substrate specificity. Smooth muscle VAP-1 was also cleaved by
O-sialoglycoprotease, suggesting that VAP-1 may contain
O-linked oligosaccharide side chains that are decorated by sialic
acids.
VAP-1 requires an intact smooth muscle microenvironment for its expression. This was evident from experiments that showed an absence of VAP-1 in primary and transformed smooth muscle cells. Endothelial VAP-1 is not as sensitive to disruption of vascular architecture, inasmuch as collagenase-digested, immunoaffinity-isolated HEV cells retain VAP-1 expression.28 Moreover, transformed endothelial cell lines and human umbilical vein endothelial cells lack all cell surface VAP-1, but nevertheless they do express low levels of intracellular VAP-1.29 These data suggest that in endothelial cells and in smooth muscle cells VAP-1 expression is differently regulated.
VCAM-1 shares similar features with VAP-1 in the expression in both
endothelial and muscle cells. The two cell types use different
transcriptional mechanisms to control VCAM-1 expression. On endothelial
cells VCAM-1 is an inducible protein,30
whereas muscle
cell VCAM-1 is ontogenically expressed on developing secondary
myotubes.8
Endothelial VCAM-1 is induced, for example,
with interleukin-1, tumor necrosis factor-, and lipopolysaccharide,
which may act via NF-
B binding sites that override the
constitutively active inhibitory element. Instead, on cultured myocytes
these transcription factors do not regulate VCAM-1 expression, because
in these cells a specific enhancer is overriding the effect of the
silencers, resulting in constitutive VCAM-1
expression.9,10
However, increased expression of VCAM-1 is
found in vivo on smooth muscle cells in atherosclerotic
plaques.31
VAP-1 on endothelial cells is also inducible in
the setting of inflammation in vivo,2
and
in vitro certain cytokines induce a modest increase in
endothelial VAP-1 expression in an organ culture model.28
Interestingly, there are also two functional NF-B elements in the
promoter of human VAP-1. In animal models endothelial VAP-1 can be
induced at the site of skin inflammation (our unpublished
observations). In contrast, the level of smooth muscle VAP-1 remains
the same during in vitro induction of tonsil with cytokines
and other proinflammatory mediators, and in vivo smooth
muscle VAP-1 induction is not seen, even in the areas of most extreme
inflammation involving smooth muscle (in the severely inflamed gut).
Furthermore, in atheromatous lesions, where induction of other
endothelial adhesion molecules like ICAM-1 and VCAM-1 is seen on smooth
muscle cells,31-33
no evidence of up-regulation of VAP-1
was seen. These data further indicate that the regulation of VAP-1
expression in endothelial cells and in smooth muscle cells is
different.
VAP-1 in smooth muscle could be envisaged to be involved in adhesion of PBLs. These mobile cells could make sequential contacts first with VAP-1 on endothelial cells and then proceed to use the same molecule for guiding migration through the smooth muscle layer. Although majority of lymphocyte trafficking takes place via HEVs, which do not have a smooth muscle layer around them, pathological accumulation of leukocytes into vessel wall in atherogenesis and vasculitis and into bronchioles in asthma clearly involves intimate contacts between leukocytes and smooth muscle cells. In vitro activated smooth muscle cells can bind activated PBLs in a VCAM-1-, ICAM-1-, and CD44-dependent manner.26,34 However, smooth muscle VAP-1 showed no appreciable PBL binding under any conditions we tested. Thus smooth muscle cells may lack some other crucial adhesion molecule(s) that is needed to stabilize VAP-1 adhesion. In the case of endothelial cells, a paradigm has arisen in which at least three sequential steps are required to secure the binding of a lymphocyte to an endothelial cell.35,36 In HEVs, VAP-1 mediates the initial stage of adhesion under conditions of shear.3 Therefore, our inability to find an adhesive function for VAP-1 on smooth muscle cells may reflect the absence of the subsequent chemokine activation or a lack of immunoglobulin superfamily members (ICAM-1, ICAM-2, and VCAM-1) on our smooth muscle cells. On the other hand, the differences in posttranslational modifications between smooth muscle cell and endothelial VAP-1 may render the smooth muscle form incapable of lymphocyte adhesion. In particular, the N-linked sugars may interfere with ligand-binding motifs of VAP-1 in smooth muscle. Finally, the most likely explanation for the lack of adhesive function for smooth muscle VAP-1 is that the plasma membrane of each smooth muscle cell is surrounded by a basement membrane. This makes a ~50-nm physical barrier which all integral membrane proteins have to penetrate before they are available for contacts with other cells. Because VAP-1 monomer is a 90-kd mushroom-shaped molecule,37 it may well be that not even the oligosaccharide residues of VAP-1 extend far enough to be seen by PBLs.
Smooth muscle VAP-1 turned out to be a semicarbazide-sensitive monoamine oxidase. The kinetic parameters of SSAO vary substantially between various species and tissues.5 However, the Km and Vmax values determined here are in close agreement with those reported earlier for SSAO in human aorta, umbilical artery, and other tissues. SSAO catalyzes oxidative deamination of primary exogenous and endogenous amines in a reaction that produces the corresponding aldehyde, hydrogen peroxide, and ammonium.5,38 The biological substrates and the function of SSAO enzymes have remained elusive. Whereas benzylamine is not believed to occur physiologically, methylamine can be formed endogenously from certain metabolic pathways and act as a possible physiological substrate for SSAO, but not for MAO, in various tissues, including vasculature. SSAO-mediated generation of aldehydes and hydrogen peroxide has been implicated in the pathogenesis of different diseases. In particular, inhibition of SSAO activity has alleviated vasculopathic changes in animal models of diabetes.39,40 Moreover, there is a role emerging for hydrogen peroxide as a signaling molecule, which regulates the expression of other adhesion molecules, cell growth, and apoptosis.41 Because smooth muscle VAP-1 is present in arterial media and in smooth muscle tumors and it is the major SSAO species in humans, it is likely to be crucial in these multiple biological functions.
In conclusion, we have shown that VAP-1 is exclusively expressed only on smooth muscle cells among different muscle cells. Smooth muscle VAP-1 has several similarities but also distinct differences in structure with the endothelial VAP-1. Most notably, the trimeric forms and N-linked posttranslational modifications of VAP-1 are only encountered on smooth muscle cells. Smooth muscle VAP-1 is not directly involved in lymphocyte binding, but instead it accounts for the most semicarbazide-sensitive monoamine oxidase activity in humans and may thus be important in the pathogenesis of multiple vasculopathies.
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Acknowledgements |
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Footnotes |
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Supported by the Finnish Academy, the Sigrid Juselius Foundation, the Finnish Cancer Union, the Paulo Foundation, and the Finnish Cultural Foundation.
The first two authors contributed equally to this work.
Accepted for publication August 5, 1999.
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1-acid glycoprotein. J Biol Chem 1982, 257:12734-12738-induced ICAM-1 and VCAM-1 expression and T lymphocyte adhesion. J Immunol 1995, 154:2358-2365[Abstract]
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