* Department of Medicine, School of Medicine, and
Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and
Cardiovascular Research Center, Massachusetts General Hospital, and
Vascular Research Division, Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts
Correspondence: Masayuki Yoshida, M.D., Dept. of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima Bldg D-621, Bunkyo-ku, Tokyo 113-8510, Japan. E-mail: masamgen{at}mri.tmd.ac.jp
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion molecule • adenovirus vector • inflammation
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF-), interleukin-1 (IL-1), or
bacterial lipopolysaccharide (LPS), peaks 4–6 h after stimulation and
rapidly decreases to basal levels [4
]. In contrast,
several studies suggested that E-selectin expression may be sustained
chronically at sites of inflammation in vivo
[5
, 6
], suggesting the differences in mRNA
stability of E-selectin in different settings [7
]. E-selectin has been shown to support the rolling of leukocytes on activated EC cells [3 ] and appears also to participate in the transition to stable adhesion that precedes transmigration [8 ]. Several studies have documented the expression of E-selectin in atherosclerotic plaque suggesting a potential role in this complex chronic inflammatory process [4 , 9 , 10 ]. However, the function(s) of E-selectin in vascular disease have remained unclear. In genetically altered mice that lack E-selectin, the possible redundant function of EC selectins made it difficult to define a precise function of E-selectin in leukocyte adhesion in vivo [11 , 12 ]. Therefore, to more critically examine the role of E-selectin in leukocyte-EC interactions in the vascular system of experimental animals, we have created a recombinant adenoviral vector containing the human E-selectin cDNA. Adenoviral vectors have been used to achieve efficient gene transfer in various types of cells, including vascular endothelium in which traditional transfection techniques are not sufficiently efficient typically to mediate expression for functional studies. Previous studies have shown that infection of a human E-selectin cDNA (AdRSVE-sel) can induce E-selectin expression in cultured human umbilical vein EC cells (HUVEC) without global activation [13 ]. Using this adenoviral vector, we were able to introduce functional E-selectin molecules into isolated rat aortic segments and then study leukocyte-EC interactions under defined flow conditions ex vivo. This model should be useful for studying E-selectin function(s) in various anatomically defined segments of the vascular system.
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Construction of recombinant adenoviral vector of human E-selectin
The construction of the recombinant adenovirus carrying
the human E-selectin cDNA under transcriptional control of the rous
sarcoma virus long terminal repeat has been described
previously in detail [19
]. AdRSVLacZ (kindly provided by
Dr. David Dichek, UCSF, San Francisco, CA) is structurally
similar to AdRSVE-sel [20
] but carries a
nuclear-targeted form of ß-galactosidase. Viral titers of purified
stocks were determined by plaque assay in 293 cells as previously
described [21
]. Several different viral stocks were used
in this study. Stocks titer ranged from 109 to
1010 pfu/ml with a particle-to-pfu ratio of
102.
Fluorescent immunobinding assay
HUVEC were plated in a 96-well culture plate and infected with
recombinant adenovirus vector; AdRSVE-sel or AdRSVLacZ was diluted in
0.05 ml DMEM + 2% FCS for 1.5 h at 37°C. Then, 0.05 ml
growth media (DMEM containing 2% FCS) was added to each well, and the
cells were incubated further for 72 h. The fluorescent immunoassay
was carried out as previously described [17
]. Briefly,
HUVEC monolayers were incubated on ice with 7A9 or Hu5/3 at a
concentration of 10 µg/ml in RPMI-1640 + 1% fetal bovine serum
(FBS) for 45 min. Plates were washed three times with RPMI-1640 +
1% FBS and then incubated with fluorescein isothiocyanate
(FITC)-conjugated goat anti-murine polyclonal immunoglobulin G (IgG)
F(ab')2 (purchased from Amersham Pharmacia Biotech,
Arlington Heights, IL) diluted 1:50 in Dulbecco’s phosphate-buffered
saline (DPBS) containing 0.9 mM CaCl2, 0.33 mM
MgCl2, on ice for 45 min. Plates were then washed twice
with DPBS + 20% FBS and twice with DPBS alone. Cells were lysed
with 0.15 ml of 0.01% NaOH in 0.1% sodium dodecyl sulfate (SDS), and
fluorescent intensity was quantified using a fluorescent plate reader
(Cytofluor II, PerSeptive Biosystems, Applied Biosystems, Foster City,
CA).
Transduction of rat aortic segment with adenovirus vector
Segments of abdominal aorta (2 cm in length) were isolated from
male Sprague-Dawley rats (bodyweight, 250 g) after anesthesia with
intraperitoneal pentobarbital (20 mg/kg). Each aortic segment was
placed in infection medium (DMEM+2% FBS) and clamped on one end. The
recombinant adenoviral vector (1x109, 1x107
pfu of AdRSVE-sel, or 1x109 pfu of a control vector
AdRSVLacZ) diluted in 100 µl PBS was introduced into the segment,
which was closed with a vascular clamp and incubated in infection
medium for 4 h at 37°C. Then clamps were released, and the
aortic segment was washed with infection medium. After an additional
72 h of incubation in infection medium at 37°C in the presence
of 5% CO2, the segment was rinsed with DMEM + 10%
FBS, and surface expression of transduced E-selectin was examined by
immunohistochemistry and western blotting using anti-E-selectin mAb
(7A9). To examine a viability of the segments, the vessel was stained
with 0.1% trypan blue for 30–45 sec before and 72 h after viral
infection and examined under a dissecting microscope. No significant
damage to the intimal EC surface of the infected segments was detected.
Western blotting
After adenovirus infection, the aortic segments were minced and
homogenized in a dounce homogenizer in the presence of 250 µl lysis
buffer [containing 20 mM benzamidine, 10 µg/ml pepstatin A, 1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, 1 mM
Na3VO4, and 0.1% Triton X-100] and incubated
on ice for 2 min. The lysates were centrifuged at 15,000 rpm for 15
min. Nonreducing sample buffer (3x) was added to the supernatant. An
aliquot (10 µg) of the samples was subjected to 8%
SDS-polyacrylamide gel electrophoresis (PAGE), transferred to a
polyvinylidene difluoride (PVDF) membrane, and incubated in 5% dry
milk in Tris buffered saline with Tween-20 (TBS-T) (20 mM Tris,
137 mM NaCl, 0.1% Tween-20, pH 7.6) at 4°C for 16 h. The
membrane was then incubated with 7A9 (10 µg/ml in 5% dry milk in
TBS-T) for 1 h, washed three times with TBS-T, and incubated with
a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG for
1 h. After washing three times with TBS-T, the protein was
detected using an enhanced chemiluminescence (ECL) kit (purchased from
Amersham).
Immunohistochemical analysis
After adenovirus infection, the aortic segments were
snapfrozen in optical cutting temperature-embedding medium
(Sakura Fine Technical Co., Tokyo) and stored at -80°C. Cryostat
sections of 5 µm thickness were cut, collected on glass slides, and
air-dried for 2 h. The slides were then fixed with 3%
paraformaldehyde in PBS for 20 min at room temperature. After extensive
washing with PBS, the slides were blocked with 1% horse serum for
1 h at room temperature in a humid atmosphere. Sections were then
incubated with a mouse anti-human E-selectin mAb (7A9), a sheep
anti-vWF polyclonal Ab, mouse anti-ICAM-1 mAb, or nonbinding control
murine IgG, all at 10 µg/ml in PBS, followed by incubation with a
biotinylated horse anti-mouse IgG (7A9, anti-rat ICAM-1 and control) or
anti-sheep IgG (anti-vWF), diluted 1:500 in PBS for 1 h. Finally,
the sections were incubated with avidin-peroxidase complex (ABC Elite
kit, Vector Labs, Burlingame, CA), visualized with an AEC Elite kit
(Vector), and observed using a light microscope (IX70, Olympus,
Tokyo, Japan).
Leukocyte adhesion assay of transduced aortic segments under
controlled flow conditions
Apparatus design
Leukocyte interaction with transduced vascular segments was
analyzed using a vessel perfusion system (Harvard Apparatus, Mills, MA)
with a slight modification. The system is composed of a plastic
chamber, a pair of cannula holders mounted to a stainless-steel bar on
the plastic platform. The vascular segment was placed in the chamber
filled with DPBS + 0.2% bovine serum albumin (BSA), and all
following procedures were carried out in the presence of this media. A
glass cannula (1 cm in length, 0.5 mm internal diameter) was introduced
into both ends of the vascular segment, tightly ligated, and then
carefully connected to the cannula holder in the apparatus. Defined
levels of flow are applied to the lumen of the vascular segment by
perfusing media (DPBS+0.2% BSA) through the system using a syringe
pump (Harvard Apparatus).
Experimental application
Adenovirally transduced aortic segments were rinsed with
perfusion media and connected to the perfusion system. Human
polymorphonuclear neutrophils (PMN) were isolated from whole blood
drawn from healthy volunteers using Lymphocyte Separating Medium (ICN
Biomedicals, Cleveland, OH) as described previously
[22
]. Isolated PMN were suspended in RPMI-1640 +
1% FBS (1x107/ml) and fluorescently labeled with
2',7’-bis(2-carboxyethyl)-5-(and -6)-carboxyfluorescein, acetoxymethyl
ester (Molecular Probes, Junction City, OR). Fluorescently
labeled PMN were then suspended in the perfusion media
(2x106/ml) and perfused in the segment with a flow rate of
0.85 ml/min (estimated wall-shear stress=1.76 dyn/cm2) for
10 min, followed by a 5-min washing-out period with the perfusion media
alone. In some experiments, THP-1 cells were used instead of PMN.
Assessment of PMN adhesion to aortic segment
The aortic segments were detached from the flow apparatus and
fixed with 2.5% glutaraldehyde in PBS for 1 h. They were then
dehydrated, dried in a critical-point dryer, coated with platinum using
a sputter coater, and examined with a scanning electron microscope. To
quantify PMN adhesion to the aortic segment, adhered PMN were collected
by flushing the segment with 1 ml of PMN-detaching media [PBS
containing 5 mM ethylenediaminetetraacetate (EDTA), 4 mM
ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic
acid (EGTA), pH 8.0], and the fluorescence intensity of the recovered
PMN was measured in a fluorescence plate reader. Comparison with the
fluorescent measurement of the lysate recovered from post-washed aortic
segments validated that 84% of adherent PMNs were recovered by the
infusion of PMN-detaching media (unpublished results). In some
experiments, 100 µl of indicated mAb (10 µg/ml in perfusion media)
was introduced into an aortic segment and incubated for 20 min at 4°C
before PMN perfusion.
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 View larger version (19K): [in a new window] |
Figure 1. Fluorescent immunoassay of HUVEC infected with AdRSVE-sel or AdRSVLacZ.
Fluorescent immunoassay of cell-surface expression of adhesion
molecules in unactivated HUVEC monolayers infected with AdRSVE-sel or
AdRSVLacZ at MOIs of 10, 50, and 100 pfu/cell compared with uninfected
HUVEC (no virus) using mAbs against E-selectin (7A9) (open bars),
ICAM-1 (Hu5/3) (solid bars; mean±SD, n=3). Data
shown are representative of three independent experiments.
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(Fig. 3C)
. To assess the effect of adenovirus gene transfer in
intimal EC adhesion-molecule expression, rat ICAM-1 expression was
investigated also. As shown in Fig. 3G
and 3H
, essentially no ICAM-1
expression was observed in the segments transduced with AdRSVE-sel and
AdRSVLacZ, suggesting the activation of the segment was minimum. In
contrast, when the segment was stimulated with rat TNF- (Fig. 3I)
,
expression of ICAM-1 was observed in the segment. vWF staining, as an
EC marker, was observed in the AdRSVE-sel-transduced (Fig. 3D)
,
AdRSVLacZ-transduced segment (Fig. 3E)
, and the TNF--stimulated
segment (Fig. 3F)
, thus documenting the preservation of the intimal EC
lining of these excised, perfused segments. Nonrelevant murine IgG did
not bind either of the segments (Fig. 3J
3K
3L)
. These data support
that E-selectin gene transfer into these excised rat aortic segments
via a recombinant adenoviral vector resulted in E-selectin protein
expression in the EC lining.
 View larger version (117K): [in a new window] |
Figure 3. Expression of transduced human E-selectin in rat aortic segments by
immunohistochemistry. Frozen sections of aortic segments transduced
with AdRSVE-sel (A, D, G, J) or AdRSVLacZ (B, E, H, K) were analyzed by
immunohistochemistry using anti-human E-selectin (A, B, C), anti-vWF
(D, E, F), anti-rat ICAM-1 (G, H, I), and nonrelevant mAb (J, K, L).
Immunoreactive human E-selectin expression was observed in
AdRSVE-sel-transduced rat aortic segments (A, arrows) but not
AdRSVLacZ-transduced aortic segments (B) or TNF--stimulated aortic
segments (C). vWF staining (D, E, F) confirmed the presence of an EC
monolayer. No staining was observed when nonrelevant mAb was used (J,
K, L). Immunoreactive rat ICAM-1 expression was observed in
TNF--stimulated aortic segments (I) but not AdRSVE-sel-transduced
rat aortic segments (G) or AdRSVLacZ-transduced aortic segments (H;
original magnification, 250x).
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. Moreover,
pretreatment of vascular segments with anti-E-selectin mAb 7A9 reduced
PMN adhesion to the transduced vascular segments significantly
(105.3±19.5, p<0.05). In contrast, pretreatment with
nonrelevant mAb (Hu5/3) did not alter PMN adhesion to the vascular
segments significantly. These data indicate that E-selectin expressed
in rat aortic segments is able to support PMN adhesion in the presence
of flow.
 View larger version (171K): [in a new window] |
Figure 4. Adhesion of PMN to excised rat aortic segments transduced with
AdRSVE-sel and AdRSVLacZ as seen by scanning electron microscope. An
aortic segment, subjected to human PMN adhesion under defined flow
condition, as described in Materials and Methods, was then fixed with
2.5% glutaraldehyde in PBS and analyzed by scanning electron
microscopy. The AdRSVE-sel-transduced segment (A) exhibited
significantly more PMN adhesion (arrows) than the control segment
transduced with AdRSVLacZ (B; original magnification: A, 450x; B,
700x).
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Figure 5. Adhesion of human PMN and THP-1 to excised, perfused, rat aortic
segments infected with AdRSVE-sel or AdRSVLacZ. The recombinant
adenoviral vector (1x109 pfu of AdRSVE-sel or a control
vector AdRSVLacZ) was introduced into the rat aortic segment. The
segment was connected to a blood perfusion system, and fluorescently
labeled human PMN (A) or a monocytic cell line THP-1 (B) were perfused
with a flow rate of 0.85 ml/min for 15 min, followed by a 5-min
wash-out period with media alone. Adhered PMN and THP-1 were collected
by incubation of the segment with 1 ml of detaching media, and the
fluorescent intensity of these samples was measured in a fluorescent
plate reader. Preincubation of the transduced segment with
anti-E-selectin mAb (7A9), but not nonrelevant mAb (Hu5/3), inhibited
PMN and THP-1 adhesion (mean±SD, n=3). Data
shown are representative of three independent experiments.
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-stimulated segment. |
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In previous in vitro studies, we discovered several important biological properties of E-selectin, for example association to EC cytoskeleton [19 ] and dephosphorylation of serine residues upon ligand binding-mediated leukocyte adhesion [23 ]. However, E-selectin function in leukocyte-EC interaction in the vascular system in vivo needs to be elucidated. Therefore, we decided to establish a more general model using the E-selectin-transduced rat aortic segment to investigate potential physiological functions of E-selectin in an intact, but ex vivo, vascular system.
Although ex vivo vessel model does not completely
reconstitute the leukocyte-EC interactions that occur in
vivo, important findings in vascular biology research were
obtained using an ex vivo system [24
,
25
]. Moreover, an isolated vessel segment was more
convenient and reproducible in an adenoviral infection procedure. The
number of EC cells in our aortic-segment model could be estimated at
105. Therefore, when we used 107 or
109 pfu to transduce in these segments, calculated MOI
could be 102 or 104, suggesting requirement of
relatively high amounts of virus to transduce aortic segments similar
to those used with in vivo studies [26
]. It
still remains difficult, however, to quantify the number of molecules
expressed on each segment, as a result of heterogeneity of
gene delivery. Nonetheless, our model will be an important intermediate
between the in vitro culture EC cell system and the in
vivo animal model system. Recent findings from another group
clearly suggest that background inflammation is a result of adenovirus
infection in rabbit model [27
]. To evaluate the function
of E-selectin-mediated leukocyte adhesion in vivo, a certain
immunoincompetent rat strain may be necessary in the future.
Previous studies have documented a correlation of PMN recruitment and inflammatory vasculitis [28 , 29 ]. However, the molecular mechanisms that mediate adhesion of PMN during these vascular diseases are not understood. Although several studies indicated enhancement of soluble adhesion-molecule expression in sera from patients with active vasculitis [30 ], a direct study of PMN adhesion to the arterial wall has not been done. Using the ex vivo aortic-segment model described here, we have demonstrated that E-selectin expressed on the luminal surface of the aortic segment was able to capture circulating PMNs. E-selectin-dependent PMN adhesion to the aortic segment may indicate an important role for this molecule during the process of inflammatory vasculitis, although we are not able to assess the possible importance of leukocyte entry from the adventitial side. It still remains unanswered, however, whether these PMNs initially captured via E-selectin can transmigrate into arterial segment or not. There may be another molecule(s) or chemokine(s) required for proper PMN recruitment. In this context, Gerszten et al. [31 ] have demonstrated recently that IL-8 and monocyte chemoattractant protein-1 (MCP-1) triggered the stable adhesion of rolling monocytes dramatically to E-selectin-transduced HUVEC in vitro. We would like to investigate the effect of these chemokines on E-selectin-mediated PMN adhesion in our ex vivo model.
Finally, we were able to show also that not only PMN but also a monocytic cell line adhered to the aortic segment overexpressing E-selectin. This indicates potential advantages of this model for the study of monocyte-EC interaction, critically important in the atherogenesis process under physiological condition.
In summary, we had expressed successfully human E-selectin in an excised, perfused rat aortic segment using an adenoviral vector. Transduced rat aortic segments were able to support E-selectin-dependent leukocyte adhesion under physiological flow conditions. This system should provide a useful tool for investigating E-selectin-dependent, leukocyte-EC cell interactions in the blood vessel.
 View larger version (73K): [in a new window] |
Figure 2. Expression of immunoreactive E-selectin protein in virally transduced
rat aortic segments. Excised segments of male Sprague-Dawley rat were
infected with AdRSVE-sel or control AdRSVLacZ viral vectors as
described in Materials and Methods. After incubation for 72 h at
37°C in the presence of 5% CO2, each segment was rinsed
with DMEM + 10% FBS, and Western blotting analysis was carried
out on lysates prepared from each type of aortic segment. A lysate
recovered from IL-1-activated HUVEC was used as a positive control
(IL-1 HUVEC). E-selectin protein expression was detected at the
predicted molecular weight in ARdSVE-sel-transduced aortic segments but
not ARdSVLacZ-transduced aortic segments (mean±SD,
n=3). Data shown are representative of three independent
experiments.
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Received December 16, 1999; revised June 1, 2000; accepted June 2, 2000.
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