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(Journal of Leukocyte Biology. 2000;68:687-692.)
© 2000 by Society for Leukocyte Biology

Adenoviral transduction of human E-selectin into isolated, perfused, rat aortic segments: an ex vivo model for studying leukocyte-endothelial interactions

Hideto Ishii*, Masayuki Yoshida{dagger}, Anthony Rosenzweig{ddagger}, Michael A. Gimbrone, Jr§, Yukio Yasukochi{dagger} and Fujio Numano*

* Department of Medicine, School of Medicine, and
{dagger} Department of Molecular Genetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan; and
{ddagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
E-selectin, a member of the selectin family of adhesion molecules, is thought to play an important role in leukocyte-endothelial (EC) interactions during inflammation and atherosclerosis. To critically examine the role of E-selectin in leukocyte-EC interactions in the vascular system, we created a recombinant adenoviral vector containing a human E-selectin cDNA (AdRSVE-sel) and examined the effect of AdRSVE-sel in an ex vivo vascular model of a rat aortic segment. A segment of abdominal aorta was isolated from a male Sprague-Dawley rat transduced with AdRSVE-sel ex vivo. After 72 h, surface expression of transduced E-selectin in the segment was confirmed by Western blotting and immunohistochemistry using anti-E-selectin mAb. Aortic segments were connected to a perfusion system and the adhesion of human polymorphonuclear neutrophils (PMN), and a human monocytic cell line (THP-1) to the EC surface was studied in the presence of a physiological level of flow (0.85 ml/min, approximate luminal surface shear stress=1.76 dyn/cm2). Adhesion of PMN was assessed by scanning electron microscopy and quantified using fluorescently labeled PMN. AdRSVE-sel transduced aortic segments mediated significantly more PMN and THP-1 adhesion than control segments transduced with AdRSVLacZ. Pretreatment of AdRSVE-sel transduced aortic segments with anti-E-selectin mAb inhibited PMN adhesion significantly, as well as THP-1. These data indicate that human E-selectin expressed in rat aortic segments can support the adhesion of human PMN as well as THP-1 under physiological flow conditions. This genetically modified, excised, vascular-segment model provides a useful tool for the study of leukocyte recruitment in the vascular system.

Key Words: adhesion molecule • adenovirus vector • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
E-selectin, a member of the selectin family of adhesion molecules, is thought to play an important role in leukocyte-endothelial (EC) interactions during acute and chronic inflammation, ischemia-reperfusion injury, and atherosclerosis [1 2 3 ]. Expression of E-selectin, observed primarily on vascular endothelium in response to inflammatory stimuli such as tumor necrosis factor {alpha} (TNF-{alpha}), 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.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
Reference stocks of the 293 cell line and the human monocytic cell line (THP-1) were obtained from American Type Culture Collection (ATCC; Rockville, MD). The 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal calf serum (FCS) [14 ], and THP-1 cells were cultured in RPMI-1640 with 10% FCS. HUVEC were isolated from umbilical cords and cultured as described previously [15 ]. Monoclonal antibodies (mAbs) used in this study were as follows: 7A9 (murine anti-human E-selectin) [16 ], Hu5/3 [murine anti-human intercellular adhesion molecule-1 (ICAM-1)] [15 ], W6/32 (murine anti-human HLA-A, -B, -C or HLA class I) [17 ], and anti-rat CD54 (murine anti-rat ICAM-1) [18 ].

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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
E-selectin expression in HUVEC transduced with AdRSVE-sel
To test the activities of the adenoviral vectors used in this study, unactivated, cultured HUVEC were infected with AdRSVE-sel or AdRSVLacZ at multiplicities of infection (MOIs) of 10, 50, and 100 pfu/cell. After 72 h, a fluorescent immunoassay using anti-E-selectin antibody was carried out. As shown in Figure 1 , there was a dose-dependent increase in the amount of E-selectin (open bars) on the surface of HUVEC transduced with AdRSVE-sel, compared with those transduced with AdRSVLacZ or uninfected HUVEC (no virus). To assess the effect of adenoviral infection on EC expression of endogenous adhesion molecules, surface expression of ICAM-1 (solid bars) was also examined. Note that there was a low level of endogenous E-selectin and ICAM-1 expression after infection with control AdRSVLacZ vector compared with uninfected HUVEC (no virus), but this was not related to the dose of the virus. These data are in agreement with our previous study [19 ]. To assess the function of the adenovirally transduced E-selectin in HUVEC, nonstatic adhesion assays were carried out using the leukocyte cell line HL60. HUVEC in C-12 tissue-culture plates were transduced with AdRSVE-sel or AdRSVLacZ at an MOI of 100 pfu/cell for 72 h. AdRSVE-sel-transduced HUVEC exhibited significantly greater HL60 adhesion (22.2±5.8% adhesion of HL60) than did AdRSVLacZ-transduced HUVEC (0.6±0.03, p<0.001). Treatment of HUVEC with anti-E-selectin mAb 7A9 (3.26±0.43, p<0.01), but not with anti-ICAM-1 mAb Hu5/3 (20.9±1.84), blocked AdRSVE-sel-dependent HL60 adhesion to HUVEC under this assay adhesion.



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  		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.

 
Introduction of AdRSVE-sel into isolated rat aortic segments
The function of E-selectin expressed in an "ex vivo" blood vessel was then evaluated using an isolated rat aortic segment. Segments of abdominal aorta were excised from male Sprague-Dawley rats, and the recombinant adenoviral vector (1x109 or 1x107 pfu of AdRSVE-sel or 1x109 pfu of a control vector AdRSVLacZ) was introduced intraluminally as described in Materials and Methods. Adenovirally transduced aortic segments were further incubated for 72 h at 37°C. The protein expression of E-selectin in these infected rat aortic segments was examined by western blotting using anti-E-selectin mAb (7A9). A large amount of immunoreactive E-selectin expression was observed in the segment transduced with 1 x 109 pfu of AdRSVE-sel but not in the segment transduced with the same pfu of AdRSVLacZ. Significantly less E-selectin immunoreactivity was detected in the segment transduced with 1 x 107 pfu of AdRSVE-sel. Immunohistochemical examination of the aortic segments transduced with 1 x 109 pfu of AdRSVE-sel or AdRSVLacZ revealed immunoreactive, human E-selectin expression in intimal EC cells (Fig. 3A , arrows) of the AdRSVE-sel-transduced segment but not in the AdRSVLacZ-transduced segment (Fig.3B) or the segment stimulated with TNF-{alpha} (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-{alpha} (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-{alpha}-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.



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  		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-{alpha}-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-{alpha}-stimulated aortic segments (I) but not AdRSVE-sel-transduced rat aortic segments (G) or AdRSVLacZ-transduced aortic segments (H; original magnification, 250x).

 
Characterization of PMN adhesion in ex vivo-perfused rat aortic segment transduced with AdRSVE-sel
After confirming human E-selectin protein expression in rat aortic segments transduced with AdRSVE-sel, we then examined the function of the transduced E-selectin molecule. Equal amounts (1x109 pfu ) of recombinant adenoviral vector (AdRSVE-sel or control AdRSVLacZ) were introduced into isolated rat aortic segments as described in Materials and Method. To study PMN adhesion to the human E-selectin expressed in the vascular wall, we have developed an ex vivo perfusion model. Freshly isolated human PMN were suspended at 2 x 106/ml in DPBS with 0.2% BSA and were perfused through the isolated segments. The average flow rate is 0.85 ml/min at an approximate vessel diameter of 1 mm. Although this flow rate is lower than that in the rat aorta in vivo, the estimated wall-shear stress of 1.76 dyn/cm2 is similar to the mean wall-shear stress calculated from magnetic resource velocity measurements of the human infrarenal abdominal aorta [22 ]. In this region of aorta, the time-averaged mean wall-shear stress that has coincided with the development of vascular disease, including atherosclerosis, is low (-1.7–1.4 dyn/cm2). Adhered PMN in the aortic segments were then analyzed by a scanning electron microscopy (SEM). As shown in Figure 4 , the AdRSVE-sel-transduced aortic segment supported significantly more PMN adhesion than did the control AdRSVLacZ-transduced segment. To quantitatively assess the leukocyte-adhesion profile of the adenoviral-transduced aortic segments, PMN were fluorescently labeled with BCECF before infusion into the cell, and the adherent population was collected subsequently by perfusion of 5 mM EDTA/4 mM EGTA in PBS (see Materials and Methods). A fluorescence intensity of the collected PMN was then measured. The AdRSVE-sel-transduced segment exhibited significantly more PMN adhesion (315.6±73.3 relative fluorescent unit) than the control segment transduced with AdRSVLacZ (31.6±7.6, p<0.05, Fig. 5A ), comparable with those activated with rat TNF-{alpha}. 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.



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  		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.

 
Because monocyte accumulation to the vessel wall is thought to be one of the earliest events in atherogenesis, and the possible involvement of E-selectin during this phenomenon has been shown [3 ], we decided to examine whether an ex vivo model of E-selectin overexpression in aortic segments can support monocyte adhesion. A monocytic cell line, THP-1 was prelabeled with BCECF and perfused to the segments transduced with AdRSVE-sel or AdRSVLacZ. As shown in Fig. 5B , the AdRSVE-sel-transduced segment, but not the AdRSVLacZ-transduced segment, supported THP-1 adhesion to a comparable degree as the TNF-{alpha}-stimulated segment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we used recombinant adenovirus vectors to investigate leukocyte adhesion to an isolated perfused rat aortic segment under physiological flow conditions. In this paper, we were able to document that this recombinant E-selectin adenovirus can also mediate intimal surface expression of E-selectin in excised rat aortic segment without including significant nonspecific activation judged by changes in ICAM-1, a sensitive marker of activation (Fig. 3) . Thus, we concluded that infection with the adenovirus vectors used in this study does not result in global EC activation.

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.



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  		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.

 

   ACKNOWLEDGEMENTS
 
The authors gratefully acknowledge the support from the Ministry of Education, Science, Sports and Culture of Japan (10178102), the Ministry of Health and Welfare of Japan. The authors wish to thank members of the Department of Obstetrics, Sanraku Hospital, Tokyo, for supplying umbilical cords, Dr. David A. Dichek for providing us adenovirus AdRSVLacZ, and Dr. Shizuko Ichinose for her help in electron-microscopy analysis.

Received December 16, 1999; revised June 1, 2000; accepted June 2, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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