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International Immunology Advance Access originally published online on October 29, 2007
International Immunology 2007 19(12):1349-1359; doi:10.1093/intimm/dxm104
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Hydrogen peroxide increases human leukocyte adhesion to porcine aortic endothelial cells via NF{kappa}B-dependent up-regulation of VCAM-1

Sukmook Lee1,2,5, Junho Chung2,4,5, In Su Ha2,5, Kyesook Yi2,5, Ji Eun Lee2, Hee Gyung Kang5, Inho Choi2,5, Kook-Hwan Oh5,6, Jae Young Kim3,5, Charles D. Surh7 and Curie Ahn2,5,6

1 Clinical Research Institute, Seoul National University Hospital, Seoul 110-744, Korea
2 Cancer Research Institute, Seoul National University, Seoul 110-744, Korea
3 Department of Biological Science, Gachon Medical School, Incheon 405-760, Korea
4 Department of Biochemistry and Molecular Biology, Seoul National University College of Medicine, Seoul 110-744, Korea
5 Transplantation Research Institute, Seoul National University, Seoul 110-744, Korea
6 Department of Internal Medicine, Seoul National University Hospital, Seoul 110-799, Republic of Korea
7 Department of Immunology, IMM-26, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

Correspondence to: C. Ahn; E-mail: curie{at}snu.ac.kr


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Although a severe shortage of organs in transplantation can be overcome by using xenotransplantation of porcine donor organs, profound immune rejection to xenogeneic antigens remains a main obstacle. To elucidate the role of hydrogen peroxide (H2O2) on xenogeneic immune responses, we investigated its effects on porcine aortic endothelial cells (PAECs). We found that H2O2 can specifically induce vascular cell adhesion molecule-1 (VCAM-1) expression on PAECs, but little on human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs). Furthermore, we further confirmed that H2O2 induces activation of NF{kappa}B in PAECs, but not in HAECs. Interestingly, cell adhesion assay showed that U937, human promonocytic leukocyte, can adhere to PAECs in an H2O2-dependent manner and by using a neutralizing assay with anti-VCAM-1-specific antibodies, we also found that the interaction is mediated primarily by VCAM-1. Finally, we also demonstrated that up-regulation of VCAM-1 expression on PAECs by reactive oxygen species-producing HL-60, human leukemic neutrophil cells, could be significantly diminished by over-expressing an H2O2-removing catalase. In summary, our results suggest that NF{kappa}B-dependent porcine VCAM-1 expression by H2O2 may promote interaction of human leukocyte to PAECs, and thus may play an important role on inducing xenogeneic immune responses.

Keywords: endothelial cells, hydrogen peroxide, pig, vascular cell adhesion molecule, xenotransplantation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 
Pig to human xenotransplantation is currently regarded as a potential alternative for organ transplantation because of the severe shortage of human donor cells, tissues and organs for transplantation. However, many obstacles still need to be overcome before xenotransplantation can become a reality (1–3). Hyperacute rejection triggered by natural antibodies can destroy the xenogeneic organ within minutes, but there is now hope that this form of rejection can be circumvented by the deletion of alpha1,3 galactosyltransferase, the enzyme responsible for the generation of bulk of the natural antibody targets (4, 5). After bypassing hyperacute rejection, vascularized xenografts are likely to encounter cell-mediated acute vascular rejection (AVR) resulting in endothelial cell injury concomitant with cytokine/chemokine release, leukocytes adhesion/infiltration and inflammation (6). Therefore, in order to prevent AVR in pig to human xenotransplantation, it has become necessary to identify participating molecules and their detailed mode of action.

Uncontrolled inflammatory responses can be particularly problematic in organ transplantation and result in graft rejection (7). Pattern of inflammation observed in AVR is generally initiated by the local accumulation of activated leukocytes (8). A cascade of events then occurs when the activated leukocytes induce a respiratory burst resulting from increased production of superoxide anion (O2–) generated by the nicotinamide adenine dinucleotide phosphate oxidase system, which then undergoes spontaneous or enzyme-catalyzed dismutation to form hydrogen peroxide (H2O2) (9). H2O2 is a small, diffusible molecule and has a longer lifespan than superoxide. Depending on its concentration, H2O2 induces the expression of immune-related molecules in inflammation via the activation of cellular signaling kinases/phosphatases and transcription factors such as NF{kappa}B and activator protein-1. Cellular injury may be also induced via conversion of H2O2 to more reactive species such as the hydroxyl radical and lipid peroxidation products (10, 11). Thus far, several major anti-oxidant enzymes such as catalase, glutathione peroxidase and peroxiredoxin (12–14) have been identified for their ability to degrade H2O2.

In a response to local inflammation, endothelial cells up-regulate various adhesion molecules involved in leukocyte infiltration into the tissues, including P- and E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), -2 and -3 and vascular cell adhesion molecule-1 (VCAM-1). Among these, VCAM-1, CD106, is known to be dominantly and inducibly expressed on endothelial cells upon activation by LPS, IL-1, INF{gamma} or tumor necrosis factor alpha (TNF{alpha}). VCAM-1 binds to very late antigen-4 (VLA-4), {alpha}4ß1 integrin, expressed on activated leukocytes in inflammation and immune rejection and plays a critical role in promoting the interaction between endothelial cells and leukocytes including monocyte and T cells (15–17). Currently, increasing attention is being paid to VCAM-1–VLA-4 interaction as targets for therapeutic interventions in inflammatory diseases. For example, M/K-2.7, a neutralizing anti-VCAM-1 mAb can reduce joint inflammation in collagen-induced arthritis (18) and small peptide antagonists of integrin {alpha}4ß1 and a {alpha}4 integrin antibody are effective in ameliorating pathology in inflammatory bowel disease, multiple sclerosis and asthma (19–21). Moreover, recent reports have shown up-regulation of VCAM-1 expression on endothelial cells to be apparent in allograft rejection (22–24). Despite these advances, the relevance of VCAM-1 in xenotransplantation is yet to be defined.

In our present study, we found that H2O2 can specifically induce VCAM-1 expression via NF{kappa}B-dependent pathway on porcine aortic endothelial cells (PAECs) and that it can also promote interaction between U937 human promonocytic leukocytes and PAECs mediated mainly by VCAM-1. Furthermore, we also demonstrated that adenovirus-mediated gene transfer of catalase, an H2O2-removing enzyme, can abrogate H2O2-induced VCAM-1 up-regulation by HL-60 on PAECs. In summary, the results obtained provide several clues for understanding a potential role of H2O2, a reactive oxygen species (ROS), in promoting human to pig xenogeneic immune response.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 Funding
 References
 
Materials
5,6-Carboxy-fluorescein succinimidyl ester (CFSE) and FITC- or Alexa Fluor-labeled goat anti-rabbit secondary antibody, rhodamine phalloidin, propidium iodide and Hoechst were obtained from Molecular Probes. HRP-conjugated antibodies to mouse or rabbit IgG and enhanced chemiluminescence were purchased from Amersham Biosciences (Uppsala, Sweden). Aprotinin, leupeptin, PFA, human tumor necrosis factor alpha (hTNF{alpha}), monophosphoryl lipid A (MPL)+ trehalose dicorynomycolate (TDM)+ cell wall skeleton (CWS) adjuvant, poly-L-lysine, hydrocortisone, insulin, endothelial cell growth supplement and H2O2 were from Sigma. Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs) and endothelial growth medium-2 bullet kit were from Cambrex (Baltimore, MD, USA). Human catalase adenovirus and anti-human catalase-specific polyclonal antibody were from Lab Frontier Inc. (Seoul, Korea). Penicillin/streptomycin, fetal bovine serum (FBS), RPMI and Dulbecco's modified Eagle's minimal essential medium were purchased from Life Technologies (Gaithersburg, MD, USA). Anti-tubulin mAb, anti-NF{kappa}B p65 antibody and anti-I{kappa}B polyclonal antibody were from Santa Cruz Inc. Commercially available anti-VCAM-1 mAb from Antigenix America Inc. (NY, USA). IkappaB kinase (IKK) inhibitor II wedelolactone was from Calbiochem. Collagenase P was from Roche. Anti-P-selectin antibodies were from AbD Serotec Inc. (Oxford, UK).

Isolation of primary PAECs
The primary PAECs were isolated from the aortas of Minnesota miniature pig generously given by Kim, Yoon Berm (Finch University of Health Sciences/Chicago Medical School, USA). With the treatment of 0.5 mg ml–1 of collagenase P at 37°C for 10 min, primary endothelial cells were collected and the cells were cultured in DMEM supplemented with 20% (v/v) FBS, 5 µM hydrocortisone, 5 µg ml–1 of insulin, 15 µg ml–1 of endothelial cell growth supplement and 1% (v/v) penicillin/streptomycin. For monitoring VCAM-1 expression by H2O2, the media was changed to DMEM supplemented with 10% FBS and 1% penicillin/streptomycin.

Establishment of PAEC lines
The PAEC lines were generated as previously reported (25). In brief, the isolated primary PAECs were obtained and then transfected with a vector containing the gene for neomycin resistance. The origin-defective SV40 genome expressing a wild-type large T antigen and the transfected cell lines were finally selected by G418.

Generation of anti-VCAM-1 polyclonal antibody
Recombinant human VCAM-1/Fc chimera (2.5 µg) was mixed in 2 ml of PBS, incubated at 37°C for 30 min, emulsified with MPL + TDM + CWS adjuvant and then injected into New Zealand white rabbits. The antibody titer of immunized rabbits was determined by ELISA using HRP-conjugated anti-rabbit IgG polyclonal antibodies as secondary antibodies. After four booster injections on a 3-week interval schedule, polyclonal sera from immunized rabbit were purified with protein G sepharose bead.

Cell culture
The PAEC lines were maintained in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. HUVECs and HAECs were maintained in EGM-2 according to the manufacturer's instruction. HL-60 human leukemia and U937 promonocytic leukocyte cell lines were cultured individually in RPMI supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin. For co-culture of PAEC lines and HL-60 cell lines, PAEC lines grown in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin were added to the different numbers of HL-60 cells and incubated for 1 day. All cells were cultured at 37°C in a humidified CO2-controlled (5%) incubator.

Treatment of H2O2 and hTNF{alpha} in human and porcine endothelial cells
In PAEC lines, H2O2 was treated for 24 h but in case of hTNF{alpha}, it was treated for 12 h in order to detect maximal expression of VCAM-1 in the cells. In HUVECs and HAECs, both H2O2 and hTNF{alpha} were treated for 24 h.

Flow cytometry
The PAEC lines, primary PAECs, HUVECs or HAECs were plated at a density of 3 x 105 cells per well in 60-mm dishes and treated by H2O2 or hTNF{alpha}, respectively, for indicated time and then the cells were trypsinized. After brief centrifugation at 1500 r.p.m. for 5 min, the pellets were washed with 1x PBS and then with blocking buffer containing 1% (w/v) BSA and 0.05% (w/v) sodium azide in 1x PBS and incubated with anti-VCAM-1 polyclonal antibody in blocking buffer adjusted to 10 µg ml–1 of final concentration at 37°C for 1 h. After centrifugation at 2000 r.p.m. for 5 min, the cells were washed with 140 µl of blocking buffer and then incubated with FITC-labeled goat anti-rabbit secondary antibody (1:100) at 37°C for 30 min. Following brief centrifugation, the pellets were washed with 140 µl of blocking buffer and then the final pellets were re-suspended with 300 µl of 2% (w/v) PFA in PBS. The VCAM-1 expression was analyzed by a flow cytometer (Beckmann Coulter, Miami, FL, USA).

Immunocytochemistry
Immunocytochemistry was performed as described previously (26). In brief, cover slips were incubated with 1 mg ml–1 of poly-L-lysine for 1 h and then PAEC lines or HAECs were grown on cover slips. After rinsing with 1x PBS two times, the cells were fixed with 3.7% (w/v) PFA for 30 min at 37°C. After washing with 1x PBS and blocking with the 1x PBS containing 5% (w/v) BSA and 0.1% Triton X-100 (TX-100) for 4 h at 4°C, the cells were incubated with 10 µg ml–1 of anti-VCAM-1 polyclonal antibody or 2 µg ml–1 of anti-NF{kappa}B antibody that recognizes NF{kappa}B p65, respectively, overnight at 4°C. The cells were washed five times with PBS containing 0.05% TX-100 and then incubated in this washing medium with Alexa Fluor-labeled goat anti-rabbit or mouse secondary antibodies (1:100), 0.1 unit per well of rhodamine phalloidin and 2 µg ml–1 of Hoechst for 1 h to visualize VCAM-1, NF{kappa}B, F-actin and nucleus, respectively. Slides were then examined under a fluorescence microscope (Olympus, Melville, NY, USA).

Infection of human catalase adenovirus
The PAEC lines (1.5 x 105 cell per well) plated on 60-mm dishes were washed once with culture medium and adenovirus was added at a multiplicity of infection (MOI) as indicated and after 2 h incubation, the culture medium was replaced with fresh medium. The cells were incubated for 1 day and then used for further experiments. In this study, the unit of MOI is IFU ml–1. IFU designates infectious unit to describe the titer of a viral suspension.

Preparation of cell extract
After harvesting porcine endothelial cells or human endothelial cells, respectively, the cells were individually lysed in 1x PBS buffer containing 1% (v/v) TX-100 and 1 mM phenylmethanesulfonyl fluoride with brief sonication. The lysed cells were centrifuged at 13 000 r.p.m. for 5 min and the supernatant was collected for further use. Protein concentrations in the extract were determined using the methods developed by Bradford (27, 28).

Immunoblot analysis
After assaying with Bradford solution, proteins were denatured by boiling for 5 min at 95°C in a Laemmli sample buffer, separated by SDS–PAGE and transferred to nitrocellulose membranes by electroblotting using the wet transfer system (Amersham Biosciences). After blocking in Tris-Tween buffered saline (TTBS) buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl and 0.05% Tween 20) containing 5% (w/v) skim milk powder, the membranes were incubated with individual mAbs or polyclonal antibodies, and then after several washings with (TTBS) buffer, the blots were incubated with anti-mouse or anti-rabbit IgG, as required, coupled with HRP. Detection was performed using an enhanced chemiluminescence kit according to the manufacturer’s instructions. To reprobe with another first antibody, membranes were incubated in striping buffer (62.5 mM Tris–HCl, pH 6.0, 100 µM 2-mercaptoethanol and 2% SDS) for 10 min at 50°C, washed and then used for further study (28).

CFSE labeling
After harvesting U937, the cells were washed two times with HBSS. The washed cells (1 x 107 cells) were incubated with CFSE solution in dimethyl sulfoxide to obtain a final concentration of 2.5 µM CFSE after resting on ice in the dark for 5 min. For quenching the labeling process, one-tenth volume of FBS was added and gently mixed for 1 min. After the brief centrifugation, the cells were re-suspended, washed two times with HBSS and counted before use.

Cell adhesion and neutralization assay
Leukocyte adhesion assays were performed with minor modification (29). Briefly, the PAEC lines plated on 60 mm dishes were stimulated with H2O2 as indicated for 24 h and then the cells were washed one time with 1x PBS containing 0.2 mM calcium chloride (CaCl2) and 0.1 mM magnesium chloride (MgCl2). Following CFSE labeling of U937 promonocytic leukocytes, 1 x 106 of the labeled cells were incubated with PAEC lines stimulated with H2O2 for 1 h at 37°C and then unbounded cells were washed five times with 1x PBS containing 0.2 mM CaCl2 and 0.1 mM MgCl2. The final cells were trypsinized and then subjected to FACS analysis. For neutralizing assay, PAEC lines stimulated with 400 µM H2O2 for 1 day were incubated with anti-VCAM-1 polyclonal antibodies as indicated for 1 h at 37°C. After five times washings with 1x PBS containing 0.2 mM CaCl2 and 0.1 mM MgCl2, the CFSE-labeled U937 cells were incubated with activated PAEC lines. Following procedures are the same as above.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 Funding
 References
 
H2O2 induces VCAM-1 expression in PAEC lines
Although the functional significance of ROS in transplantation is being increasingly appreciated (41–45), the potential role of H2O2 in pig to human xenotransplantation is largely unknown. In order to investigate the role of H2O2 on xenogeneic immune responses, PAEC lines were treated with H2O2 and analyzed for activation of PAECs by monitoring the up-regulation of VCAM-1 expression, a well-known endothelial cell marker. As shown in Fig. 1(A), exposure to H2O2 readily induced up-regulation of VCAM-1 expression on PAEC lines, which normally express low levels of VCAM-1. H2O2 induced VCAM-1 expression in a concentration- and a time-dependent manner with detectable levels of VCAM-1 up-regulation from 50 µM of H2O2 for a few hours (Fig. 1B and C). The up-regulation of VCAM-1 was detectable by both FACS analysis and by immunocytochemistry (Fig. 1A and D). These results suggest that H2O2 could be a critical molecule in inducing PAEC activation and VCAM-1 up-regulation.


Figure 1
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  		Fig. 1. Effects of H2O2 on up-regulation of VCAM-1 expression on PAEC lines. (A) PAEC lines were cultured in the absence (thin line) or presence (thick line) of 400 µM H2O2 for 24 h and stained for VCAM-1 expression using a rabbit polyclonal antibody followed by FITC-conjugated anti-rabbit IgG antibody as described in Methods. The dotted line represents PAECs staining with only the secondary antibody. (B) PAEC lines were incubated with increasing concentrations of H2O2 for 24 h and then stained for VCAM-1 as in (A). (C) PAEC lines were incubated with 400 µM H2O2 for increasing time and then stained for VCAM-1 expression as in (A). (D) PAEC lines grown on cover slip were cultured in the absence or presence of 400 µM H2O2 for 1 day, fixed and then stained with the anti-VCAM-1 antibody or rhodamine phalloidin for detecting F-actin. The bound antibodies were detected with Alexa Fluor-conjugated goat anti-rabbit IgG. NT means ‘no treatment’. The results shown are representative of at least two separate experiments. Significant differences from control samples (P < 0. 05) according to the Student’s t-test are indicated by #.

 
H2O2 induces strong up-regulation of VCAM-1 expression on pig but not on human endothelial cells
It is well known that human endothelial cells up-regulate VCAM-1 expression when cultured with hTNF{alpha}, which is one of the strongest inducers of the expression of cell adhesion molecules including VCAM-1 (30, 31). Interestingly, hTNF{alpha} also increased expression of VCAM-1 on PAEC lines, but this up-regulation peaked at 12 h after exposure and declined at 24 h after its treatment (Fig. 2A). We then compared the responsiveness to H2O2 and hTNF{alpha}. In contrast to PAEC lines, two primary human endothelial cells, HUVEC and HAEC, did not up-regulate VCAM-1 expression when exposed to H2O2 (Fig. 2B). These results indicate that both H2O2 and hTNF{alpha} can elevate VCAM-1 expression in PAEC lines, whereas only hTNF{alpha} can increase expression of VCAM-1 on HUVEC and HAEC. Hence, H2O2 could be a key agent in specifically priming pig tissues for xenogeneic immune responses.


Figure 2
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  		Fig. 2. Strong up-regulation of H2O2-induced VCAM-1 expression on PAEC lines but not on HUVECs and HAECs. (A) PAEC lines were incubated with 20 ng ml–1 of hTNF{alpha} for the indicated time periods and VCAM-1 expression detected as described in Fig. 1. (B) PAEC lines, HUVECs and HAECs were cultured in the absence (white) or presence of 400 µM H2O2 (black) or 20 ng ml–1 of hTNF{alpha} (gray) and then stained for VCAM-1 expression as described in Fig. 1. The results shown are representative of three separate experiments performed in duplicates. Significant differences from control samples (P < 0. 05) according to the Student’s t-test are indicated by #. + denotes P = 0.051.

 
H2O2 specifically induces NF{kappa}B activation in pig but not in human endothelial cells
Nuclear factor kappaB (NF{kappa}B) is a transcription factor that controls the expression of a number of genes important in mediating immune and inflammatory responses. In quiescent cells, NF{kappa}B is in an inactivated state complexed with I{kappa}B in the cytosol, and upon cell activation, NF{kappa}B is translocated into the nucleus after I{kappa}B degradation (32, 33). To investigate whether H2O2 can activate NF{kappa}B in PAEC lines and HAECs, these cells were incubated with increasing concentrations of H2O2 for 24 h and degradation of I{kappa}B analyzed; hTNF{alpha} was used as a positive control to observe I{kappa}B degradation. As shown in Fig. 3(A) (left), exposure to H2O2 induced I{kappa}B degradation in PAEC lines, similar to that observed with hTNF{alpha}. Unlike PAEC lines, treatment with H2O2 did not induce I{kappa}B degradation in HAECs, even though I{kappa}B degradation in these cells was observed after exposure to hTNF{alpha} (Fig. 3A, right). To further verify NF{kappa}B activation by H2O2 in PAEC lines, we then performed immunocytochemistry. Figure 3(B) shows that H2O2 and hTNF{alpha} induced NF{kappa}B translocation from the cytosol into the nucleus in PAEC lines whereas H2O2-induced translocation of NF{kappa}B did not occur in HAECs; control HAECs treated with hTNF{alpha} displayed NF{kappa}B translocation. In order to further clarify the relationship between NF{kappa}B activation and H2O2-induced VCAM-1 expression on PAEC lines, we pre-treated IKK, an upstream kinase regulating NF{kappa}B activation, inhibitor wedelolactone and checked its effect on H2O2-induced VCAM-1 expression on PAEC lines. As shown in Fig. 3(C), we observed that it could significantly inhibit H2O2-induced VCAM-1 expression in the cells. These results suggest that NF{kappa}B activation may play a key role in H2O2-dependent VCAM-1 up-regulation on PAECs.


Figure 3
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  		Fig. 3. Effects of H2O2 and hTNF{alpha} on NF{kappa}B activation in PAEC lines and HAECs. (A) PAEC lines and HAECs were incubated with indicated concentrations of H2O2 or hTNF{alpha} for 24 h and then subjected to immunoblotting with anti-I{kappa}B-specific polyclonal antibodies as described in Methods (upper panel). The detection of ß-tubulin was used as loading control. The band intensity of I{kappa}B normalized by ß-tubulin was calculated as % of control and then depicted as graph bar (lower panel). (B) PAEC lines and HAECs grown on cover slips were incubated with 200 µM H2O2 or 20 ng ml–1 hTNF{alpha} and then immunocytochemistry with antibody against NF{kappa}B p65 was performed to detect NF{kappa}B translocation from cytosol to nucleus as described in Methods. The arrows designate cells in which nuclear translocation of NF{kappa}B p65 is considered to be occurred. (C) After pre-treating indicated concentrations of IKK inhibitor wedelolactone on PAEC lines, the cells were incubated in the absence or presence of 200 µM H2O2 for 24 h and then performed flow cytometry as described in Methods. The results shown are representative of at least two separate experiments.

 
H2O2 augments VCAM-1-mediated adhesion of U937 human promonocytic leukocytes to PAEC lines
To determine whether exposure to H2O2 may promote leukocyte adhesion to pig endothelial cells, we first checked adhesion of U937, human promonocytic leukocytes, to PAEC lines treated with H2O2. PAEC lines previously exposed to H2O2 for 24 h were cultured for 1 h with CFSE-labeled U937 cells and then the proportion of bound U937 cells to PAEC lines was analyzed by flow cytometry. As shown in Fig. 4(A and B), more U937 cells were bound significantly on H2O2-treated PAEC lines than on untreated PAEC lines. To determine whether the enhanced binding of U937 cells to H2O2-treated PAECs was mediated by VCAM-1, the binding assay was performed in the presence of increasing concentrations of both anti-VCAM-1 polyclonal antibodies that we generated and commercially available anti-VCAM-1 mAbs. We found that anti-VCAM-1 antibodies significantly reduced the interaction between U937 cells and activated PAEC lines (Fig. 4B). These results suggest that H2O2-induced up-regulation of porcine VCAM-1 may augment adhesion of human leukocytes to porcine endothelial cells.


Figure 4
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  		Fig. 4. Effects of H2O2 on VCAM-1-mediated adhesion of U937 human promonocytic leukocytes to PAEC lines. (A) PAEC lines cultured in the absence (thin line) or presence (thick line) of 400 µM H2O2 for 24 h were incubated with CFSE-labeled U937 promocytic leukocytes for 1 h. After washing the cells gently for five times, the cells were subjected to flow cytometry as described in Methods. The dotted line representing CFSE-labeled U937 cells is shown as a positive control. (B) Adhesion assay of CSFE-labeled U937 cells was performed with PAEC lines cultured in the absence (white) or presence (black) of 400 µM H2O2 for 24 h. The % values of bound CFSE-labeled U937 cells are depicted as vertical bars. (C) CFSE-labeled U937 cells are incubated with H2O2-activated PAEC lines in the presence of increasing concentrations of polyclonal anti-VCAM-1 antibodies and then the bound U937 cells analyzed as described in Methods. Asterisk denotes the neutralizing effect by a commercially available anti-VCAM-1-specific mAb. The results shown represent the means ± SDs obtained from the representative of three separate experiments performed in duplicates. Significant differences from control samples (P > 0. 05) according to the Student’s t-test are indicated by #.

 
VCAM-1 up-regulation by HL-60 on PAECs could be abrogated by the over-expression of H2O2-removing catalase
To determine whether ROS produced during xenogeneic immune responses can induce up-regulation of VCAM-1 on PAEC, PAEC lines were cultured with increasing numbers of HL-60 human leukemic neutrophil cell line for 24 h and then checked for VCAM-1 expression on PAEC lines. As shown in Fig. 5(A and B), we observed increasing levels of VCAM-1 up-regulation on PAEC lines with higher numbers of HL-60 cells. We also confirmed that HL-60 itself does not express VCAM-1 (data not shown). In order to investigate the role of H2O2 produced by HL-60 cells in inducing up-regulation of VCAM-1 on PAECs, PAEC lines were infected with adenovirus-encoding catalase, an anti-oxidant enzyme which catalyzes H2O2 into H2O and O2. Expression of catalase in PAEC lines was induced with adenovirus in a viral titer-dependent manner and was readily detectable by immunoblotting with anti-catalase-specific polyclonal antibody (Fig. 5C). Expression of catalase significantly abrogated the up-regulation of VCAM-1 on PAEC lines induced by HL-60 cells (Fig. 5D). In addition, we also verified that the infection with catalase-encoding adenovirus did not affect VCAM-1 up-regulation on PAEC lines even though we treated at MOI 100 of the adenovirus to the cells (data not shown). This finding suggests that H2O2 produced by human neutrophils may play a key role in inducing VCAM-1 up-regulation on pig endothelial cells.


Figure 5
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  		Fig. 5. Effects of H2O2 produced by HL-60 on up-regulation of VCAM-1 expression on PAEC lines. (A) Increasing numbers of HL-60 cells were incubated with PAEC lines for 1 day and then VCAM-1 expression detected as described in Methods. Dotted line represents staining on PAEC lines detected with only the secondary antibody. (B) The result shown in (A) was re-plotted as vertical bar. (C) PAEC lines were infected with increasing MOI of catalase-encoding adenovirus for 1 day and then the harvested cells were subjected to immunoblotting with anti-catalase-specific polyclonal antibodies as described in Methods. The detection of ß-tubulin was used as loading control. (D) PAEC lines were incubated with MOI 20 of catalase adenovirus for 1 day and then incubated with HL-60 for additional 24 h and then VCAM-1 expression on PAEC lines analyzed as described in Methods. Results shown are representative of three separate experiments. Significant differences from control samples (P > 0. 05) according to the Student’s t-test are indicated by #.

 
H2O2 up-regulates NF{kappa}B-dependent VCAM-1 expression on freshly isolated primary PAECs
In order to verify whether H2O2 specifically up-regulates NF{kappa}B-dependent VCAM-1 expression on primary PAECs, we first isolated primary PAECs from the aortas of Minnesota miniature pig as described in Methods. Then, we compared the effect of H2O2 on PAEC lines (data not shown) and primary PAECs with that on HUVEC in the same media condition (DMEM + 10% FBS) and performed flow cytometry. As shown in Fig. 6(A), we observed that H2O2 specifically up-regulates VCAM-1 expression on primary PAECs but not on HUVECs, showing similar results on PAEC lines. Furthermore, to further clarify the relationship between NF{kappa}B activation and H2O2-induced VCAM-1 expression on primary PAECs, we pre-treated IKK inhibitor wedelolactone to the cells in the absence or presence of H2O2 and then performed flow cytometry. As shown in Fig. 6(B), we confirmed that NF{kappa}B is also critical for up-regulating VCAM-1 expression by H2O2 on primary PAECs. In summary, these findings suggest that the results shown with PAEC lines are almost similar to those with primary PAECs.


Figure 6
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  		Fig. 6. The effect of H2O2 on NF{kappa}B-dependent VCAM-1 expression on freshly isolated primary PAECs. (A) HUVECs and freshly isolated primary PAECs were incubated in the absence (thin line) or presence (thick line) of 400 µM H2O2 for 24 h, respectively, in DMEM supplemented with 10% FBS and then flow cytometry was performed. (B) After pre-treating 10 nM of IKK inhibitor wedelolactone, freshly isolated primary PAECs was incubated in the absence or presence of 400 µM H2O2 for 24 h and the cells were subjected to flow cytometry as described in Methods. Results shown are representative of at least two separate experiments. Significant differences from control samples (P > 0. 05) according to the Student’s t-test are indicated by #.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 Funding
 References
 
In contrast, the recent advances toward potentially overcoming antibody-mediated hyperacute rejection in pig to human xenotransplantation progress to minimize the subsequent cell-mediated AVR of xenografts has been negligible. To this end, we focused on elucidating the molecular mechanism of the initial activation of pig endothelial cells in xenogeneic immune responses and have found that H2O2 itself induces NF{kappa}B-dependent VCAM-1 expression in PAECs, which can promote interaction of human leukocytes to PAECs. Furthermore, adenovirus-delivered transfection of an anti-oxidant enzyme catalase is described as a potential approach for controlling xenogeneic immune response induced by activated leukocytes.

We report for the first time that H2O2 specifically induces NF{kappa}B-dependent VCAM-1 expression on PAECs and promotes binding of human leukocytes to PAECs. Several lines of evidence supported this notion: First, H2O2 was found to induce significant up-regulation of VCAM-1 expression on PAECs. Second, HL-60 human cell line was found to generate ROS and increase VCAM-1 expression on PAECs in an H2O2-depedent manner. Third, H2O2 was also found to induce I{kappa}B degradation and NF{kappa}B translocation to nucleus in PAECs. Fourth, IKK inhibitor wedelolactone showed a significant inhibition of H2O2-dependent VCAM-1 expression on PAECs. Fifth, H2O2 was found to increase the interaction between U937 human promonocytic leukocytes and PAECs and through neutralizing assay with anti-VCAM-1-specific antibodies, H2O2-dependent VCAM-1 was also found to be crucial for the interaction between PAECs and human leukocytes. Taken together, our results lead us to the conclusion that H2O2 may play a key role in immune response of human leukocyte toward porcine endothelial cells by inducing VCAM-1 up-regulation, thereby promoting or sustaining xenogeneic immune responses in pig to human xenotransplantation.

In this study, we have used 200 or 400 µM of H2O2 to activate PAEC lines as well as primary PAECs in vitro. Not much is known regarding the precise range of H2O2 concentrations that are produced locally during inflammation under in vivo conditions. Hence, we based our working concentrations from a survey of previously published relevant papers. Based on our surveys, 0.1–1 mM of H2O2 has been used to activate human endothelial cells (34–36). The concentration is almost similar to H2O2 concentration we used in our experimental setting.

In the present study, a moderate dose (400 µM) of H2O2 seems to exclusively up-regulate VCAM-1 expression on PAECs but not on HUVECs and HAECs, suggesting that porcine endothelial cells might be more sensitive to H2O2 for VCAM-1 expression. Although we checked H2O2-induced up-regulation of VCAM-1 on PAEC lines (data not shown), primary PAECs and HUVECs even in the same media condition, we also obtained the similar results (Fig. 6A). Furthermore, with this dose of H2O2, we did not observe its additive effect on up-regulating VCAM-1 expression on HUVECs in response to hTNF{alpha}, also showing the intrinsic property of HUVECs less sensitive to H2O2 (Supplementary Figure 3, available at International Immunology Online). However, at a high dose (>1000 µM) of H2O2, we also found that HUVECs can significantly up-regulate VCAM-1 expression (Supplementary Figure 1, available at International Immunology Online). Based on previous reports, it has been known that superoxide or nitric oxide participates in VCAM-1 expression in human endothelial cells (37–41). This finding suggests that in porcine endothelial cells, H2O2 may be more crucial for inducing VCAM-1 up-regulation but, in contrast to human endothelial cells, other factors may be important for VCAM-1 expression. However, we cannot exclude the possibility that H2O2 may participate in elevating molecules involved in immune responses other than VCAM-1 expression on human endothelial cells because our finding is just focused on its effect on VCAM-1 expression on human endothelial cells as these were not examined.

In inflammatory responses, it has been assumed that injured endothelial cells secrete various cytokines and chemokines in order to recruit leukocytes, including monocyte/macrophages and T cells that mediate endothelial cell death (42, 43). However, the molecules promoting the recruitment and infiltration of leukocytes to endothelial cells have not been clearly identified. Our findings that H2O2 induces VCAM-1 expression on PAECs and that the increased adhesion of human leukocyte to the endothelial cells is mediated by H2O2-induced VCAM-1 support the notion that H2O2-induced porcine VCAM-1 may be a critical target molecule that may initiate xenogeneic immune responses in pig to human xenotransplantation. Furthermore, this hypothesis is supported by several lines of evidence showing that VCAM-1 is essential for the interaction of endothelial cells with T cells and for transmigration of monocyte/macrophages (44–46). Thus, we suggest that disrupting the interaction of leukocytes with injured endothelial cells by suppressing porcine VCAM-1 expression could be a viable approach to prevent xenogeneic immune responses. However, knockout model of mouse VCAM-1 was found to show embryonic lethality in mice (47). For this reason, a new alternative for the regulation of this protein is still required.

NF{kappa}B is a known reduction/oxidation (redox)-sensitive transcription factor and plays a central role in activating the immune responses and inflammation through regulation of gene expression of a large number of cytokines and other immune response genes (32). More specifically, in human cells, NF{kappa}B activation was reported to be critical for expressing cell adhesion molecules such as E- and P-selectin, ICAM-1 and VCAM-1 (48). Thus, in this study, we monitored VCAM-1, one of inflammatory molecules expressing on endothelial cells via NF{kappa}B activation, as an index of NF{kappa}B function. We found that H2O2-dependent VCAM-1 expression occurs on PAECs but not on human endothelial cells. Hence, this suggests that the expression of porcine VCAM-1 may be dependent on NF{kappa}B activation. The notion is further supported by our results: First, H2O2 increased I{kappa}B degradation on PAECs but not on HAECs. Second, H2O2 induced NF{kappa}B translocation from cytosol to nucleus on PAECs but not on HAECs. Third, most importantly, IKK inhibitor wedelolactone significantly inhibited VCAM-1 expression by H2O2 on PAECs. Fourth, on PAECs, H2O2 also up-regulated the expression of P-selectin and ICAM-1, all of which are known to be expressed by NF{kappa}B activation whereas on HUVECs, it affected little to up-regulate the expression of P-selectin and ICAM-1 (Supplementary Figure 2, available at International Immunology Online). In summary, these results lead us to the speculation that NF{kappa}B activation may be a prerequisite for H2O2-induced VCAM-1 up-regulation on PAECs.

Controlling ROS level produced in allotransplantation may be critical for minimizing immune responses in transplantation. In previous reports, the administration of several anti-oxidants such as metallothionein, carvedilol, ascorbic acid, alpha-tocopherol and N-acetylcysteine has been shown to exert a protective effect against allograft rejection (49–52). Furthermore, pyruvate, as an energetic substrate, is a potentially beneficial nutrient for patients undergoing organ transplantation by removing ROS (53). In this study, we have shown that adenovirus-mediated gene transfer of catalase, an H2O2-removing enzyme, to PAECs abrogated H2O2-induced VCAM-1 expression in PAECs. This approach will provide a basic clue for producing transgenic pig, a source of genetically modified donor organ. Taken together, these results suggest that over-expressing catalase using adenovirus infection to donor cells or tissues may protect porcine solid organ from damages induced by H2O2 in pig to human xenotransplantation.

In summary, we have demonstrated that H2O2 induces NF{kappa}B-dependent VCAM-1 expression in PAECs and promotes adhesion of human leukocytes. Furthermore, catalase over-expression by transgenic expression may be a viable therapy for preventing xenogeneic immune responses in xenograft. We suggest that in pig to human xenotransplantation, the endothelium of the porcine donor organ injured initially by trauma or other stimuli may be further activated by intracellular or extracellular H2O2 produced by locally accumulated leukocytes adjacent to the endothelium. This then increases NF{kappa}B-dependent VCAM-1 expression, leukocyte adhesion to PAECs, and acts as a positive feedback mechanism to intensify further immune responses ultimately leading to the induction of endothelial cell death. Thus, as a preventive way for alleviating the xenogeneic immune responses, over-expressing catalase in target endothelial cells may be applicable. In near future, we hope to study the details and fine-tuning of the roles of anti-oxidant enzymes in animal model representing pig xenograft.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
Funding
 References
 
Supplementary Figures 1–3 are available at International Immunology Online.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
References
 
National R&D Program of The Ministry of Science and Technology in the Republic of Korea (F104AD010006-06A0401-00610 and F104AD010002-06A0401-00210); Regenomics Program of Korea Science and Engineering Foundation (2006-01576).


   Acknowledgements
 
The authors thank Mauro S. Sandrin (The University of Melbourne, Victoria, Australia) for a generous advice to this work, Hur, Young Mi for valuable help in the generation of anti-VCAM-1 polyclonal antibody and Kim, Donghee for advice in part in the characterization of leukocyte adhesion experiment.


   Abbreviations
 
AVR, acute vascular rejection
CaCl2, calcium chloride
CFSE, 5,6-carboxy-fluorescein succinimidyl ester
CWS, cell wall skeleton
FBS, fetal bovine serum
HAEC, human aortic endothelial cell
hTNF{alpha}, human tumor necrosis factor alpha
HUVEC, human umbilical vein endothelial cell
H2O2, hydrogen peroxide
ICAM-1, intercellular cell adhesion molecule-1
IKK, ikappaB kinase
MgCl2, magnesium chloride
MOI, multiplicity of infection
MPL, monophosphoryl lipid A
NF{kappa}B, nuclear factor kappaB
PAEC, porcine aortic endothelial cell
ROS, reactive oxygen species
TDM, trehalose dicorynomycolate
TNF{alpha}, tumor necrosis factor alpha
TX-100, Triton X-100
VCAM-1, vascular cell adhesion molecule-1
VLA-4, very late antigen-4

   Notes
 
Transmitting editor: S. Hedrick

Received 24 January 2007, accepted 27 September 2007.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 Funding
 References
 

  1. Cascalho M, Platt JL. Xenotransplantation and other means of organ replacement. Nat. Rev. Immunol. (2001) 1:154.[CrossRef][Medline]
  2. Cascalho M, Platt JL. The immunological barrier to xenotransplantation. Immunity (2001) 14:437.[CrossRef][Web of Science][Medline]
  3. Sachs DH, Colvin RB, Cosimi AB, et al. Xenotransplantation—caution, but no moratorium [comment]. Nat. Med. (1998) 4:372.[Web of Science][Medline]
  4. Kuwaki K, Tseng YL, Dor FJ, et al. Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat. Med. (2005) 11:29.[CrossRef][Web of Science][Medline]
  5. Yamada K, Yazawa K, Shimizu A, et al. Marked prolongation of porcine renal xenograft survival in baboons through the use of alpha1,3-galactosyltransferase gene-knockout donors and the cotransplantation of vascularized thymic tissue. Nat. Med. (2005) 11:32.[CrossRef][Web of Science][Medline]
  6. Auchincloss H Jr, Sachs DH. Xenogeneic transplantation. Annu. Rev. Immunol. (1998) 16:433.[CrossRef][Web of Science][Medline]
  7. Bach FH, Ferran C, Soares M, et al. Modification of vascular responses in xenotransplantation: inflammation and apoptosis [see comment]. Nat. Med. (1997) 3:944.[CrossRef][Web of Science][Medline]
  8. Holgersson J, Ehrnfelt C, Hauzenberger E, Serrander L. Leukocyte endothelial cell interactions in pig to human organ xenograft rejection. Vet. Immunol. Immunopathol. (2002) 87:407.[CrossRef][Web of Science][Medline]
  9. Rosen GM, Pou S, Ramos CL, Cohen MS, Britigan BE. Free radicals and phagocytic cells. FASEB J. (1995) 9:200.[Abstract]
  10. Finkel T. Oxygen radicals and signaling. Curr. Opin. Cell Biol. (1998) 10:248.[CrossRef][Web of Science][Medline]
  11. Rhee SG. Redox signaling: hydrogen peroxide as intracellular messenger. Exp. Mol. Med. (1999) 31:53.[Web of Science][Medline]
  12. Arthur JR. The glutathione peroxidases. Cell. Mol. Life Sci. (2000) 57:1825.[CrossRef][Web of Science][Medline]
  13. Calabrese EJ, Canada AT. Catalase: its role in xenobiotic detoxification. Pharmacol. Ther. (1989) 44:297.[CrossRef][Web of Science][Medline]
  14. Rhee SG, Kang SW, Netto LE, Seo MS, Stadtman ER. A family of novel peroxidases, peroxiredoxins. Biofactors (1999) 10:207.[Web of Science][Medline]
  15. Foster CA. VCAM-1/alpha 4-integrin adhesion pathway: therapeutic target for allergic inflammatory disorders. J. Allergy Clin. Immunol. (1996) 98:S270.[CrossRef][Web of Science][Medline]
  16. Yusuf-Makagiansar H, Anderson ME, Yakovleva TV, Murray JS, Siahaan TJ. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases. Med. Res. Rev. (2002) 22:146.[CrossRef][Web of Science][Medline]
  17. Jackson DY. Alpha 4 integrin antagonists. Curr. Pharm. Des. (2002) 8:1229.[CrossRef][Web of Science][Medline]
  18. Carter RA, Campbell IK, O'Donnel KL, Wicks IP. Vascular cell adhesion molecule-1 (VCAM-1) blockade in collagen-induced arthritis reduces joint involvement and alters B cell trafficking. Clin. Exp. Immunol. (2002) 128:44.[CrossRef][Web of Science][Medline]
  19. Miller DH, Khan OA, Sheremata WA, et al. A controlled trial of natalizumab for relapsing multiple sclerosis [see comment]. New Engl. J. Med. (2003) 348:15.[Abstract/Free Full Text]
  20. Ghosh S, Goldin E, Gordon FH, et al. Natalizumab for active Crohn's disease [see comment]. New Engl. J. Med. (2003) 348:24.[Abstract/Free Full Text]
  21. Kudlacz E, Whitney C, Andresen C, et al. Pulmonary eosinophilia in a murine model of allergic inflammation is attenuated by small molecule alpha4beta1 antagonists. J. Pharmacol. Exp. Ther. (2002) 301:747.[Abstract/Free Full Text]
  22. Denton MD, Davis SF, Baum MA, et al. The role of the graft endothelium in transplant rejection: evidence that endothelial activation may serve as a clinical marker for the development of chronic rejection. Pediatr. Transplant. (2000) 4:252.[CrossRef][Medline]
  23. Kamoun M. Cellular and molecular parameters in human renal allograft rejection. Clin. Biochem. (2001) 34:29.[CrossRef][Web of Science][Medline]
  24. Rothman A, Mann D, Behling CA, et al. Increased expression of endoarterial vascular cell adhesion molecule-1 mRNA in an experimental model of lung transplant rejection: diagnosis by pulmonary arterial biopsy. Transplantation (2003) 75:960.[CrossRef][Web of Science][Medline]
  25. Kim D, Kim JY, Koh HS, et al. Establishment and characterization of endothelial cell lines from the aorta of miniature pig for the study of xenotransplantation. Cell Biol. Int. (2005) 29:638.[CrossRef][Web of Science][Medline]
  26. Lee S, Park JB, Kim JH, et al. Actin directly interacts with phospholipase D, inhibiting its activity. J. Biol. Chem. (2001) 276:28252.[Abstract/Free Full Text]
  27. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (1976) 72:248.[CrossRef][Web of Science][Medline]
  28. Lee S, Kim JH, Lee CS, et al. Collapsin response mediator protein-2 inhibits neuronal phospholipase D(2) activity by direct interaction. J. Biol. Chem. (2002) 277:6542.[Abstract/Free Full Text]
  29. Spertini O, Luscinskas FW, Kansas GS, et al. Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion. J. Immunol. (1991) 147:2565.[Abstract/Free Full Text]
  30. Raab M, Daxecker H, Markovic S, Karimi A, Griesmacher A, Mueller MM. Variation of adhesion molecule expression on human umbilical vein endothelial cells upon multiple cytokine application. Clin. Chim. Acta. (2002) 321:11.[Web of Science][Medline]
  31. Rahman A, Kefer J, Bando M, Niles WD, Malik AB. E-selectin expression in human endothelial cells by TNF-alpha-induced oxidant generation and NF-kappaB activation. Am. J. Physiol. (1998) 275:L533.[Web of Science][Medline]
  32. Karin M, Greten FR. NF-kappaB: linking inflammation and immunity to cancer development and progression. Nat. Rev. Immunol. (2005) 5:749.[CrossRef][Web of Science][Medline]
  33. Kabe Y, Ando K, Hirao S, Yoshida M, Handa H. Redox regulation of NF-kappaB activation: distinct redox regulation between the cytoplasm and the nucleus. Antioxid. Redox Signal. (2005) 7:395.[CrossRef][Web of Science][Medline]
  34. Okayama N, Coe L, Oshima T, Itoh M, Alexander JS. Intracellular mechanisms of hydrogen peroxide-mediated neutrophil adherence to cultured human endothelial cells. Microvasc. Res. (1999) 57:63.[CrossRef][Web of Science][Medline]
  35. Suhara T, Fukuo K, Sugimoto T, et al. Hydrogen peroxide induces up-regulation of Fas in human endothelial cells. J. Immunol. (1998) 160:4042.[Abstract/Free Full Text]
  36. Men doza L, Carrascal T, De Luca M, et al. Hydrogen peroxide mediates vascular cell adhesion molecule-1 expression from interleukin-18-activated hepatic sinusoidal endothelium: implications for circulating cancer cell arrest in the murine liver. Hepatology (2001) 34:298.[CrossRef][Web of Science][Medline]
  37. ten Kate M, van der Wal JB, Sluiter W, et al. The role of superoxide anions in the development of distant tumour recurrence. Br. J. Cancer. (2006) 95:1497.[CrossRef][Web of Science][Medline]
  38. Lin SJ, Shyue SK, Hung YY, et al. Superoxide dismutase inhibits the expression of vascular cell adhesion molecule-1 and intracellular cell adhesion molecule-1 induced by tumor necrosis factor-alpha in human endothelial cells through the JNK/p38 pathways. Arterioscler. Thromb. Vasc. Biol. (2005) 25:334.[Abstract/Free Full Text]
  39. Chen XL, Zhang Q, Zhao R, Ding X, Tummala PE, Medford RM. Rac1 and superoxide are required for the expression of cell adhesion molecules induced by tumor necrosis factor-alpha in endothelial cells. J. Pharmacol. Exp. Ther. (2003) 305:573.[Abstract/Free Full Text]
  40. Cartwright JE, Whitley GS, Johnstone AP. Endothelial cell adhesion molecule expression and lymphocyte adhesion to endothelial cells: effect of nitric oxide. Exp. Cell Res. (1997) 235:431.[CrossRef][Web of Science][Medline]
  41. Tsao PS, Buitrago R, Chan JR, Cooke JP. Fluid flow inhibits endothelial adhesiveness. Nitric oxide and transcriptional regulation of VCAM-1. Circulation (1996) 94:1682.[Abstract/Free Full Text]
  42. Kofler S, Nickel T, Weis M. Role of cytokines in cardiovascular diseases: a focus on endothelial responses to inflammation. Clin. Sci. (2005) 108:205.[CrossRef][Web of Science][Medline]
  43. Kluger MS. Vascular endothelial cell adhesion and signaling during leukocyte recruitment. Adv. Dermatol. (2004) 20:163.[Medline]
  44. Mueller JP, Evans MJ, Cofiell R, Rother RP, Matis LA, Elliott EA. Porcine vascular cell adhesion molecule (VCAM) mediates endothelial cell adhesion to human T cells. Development of blocking antibodies specific for porcine VCAM. Transplantation (1995) 60:1299.[Web of Science][Medline]
  45. Nakajima H, Sano H, Nishimura T, Yoshida S, Iwamoto I. Role of vascular cell adhesion molecule 1/very late activation antigen 4 and intercellular adhesion molecule 1/lymphocyte function-associated antigen 1 interactions in antigen-induced eosinophil and T cell recruitment into the tissue. J. Exp. Med. (1994) 179:1145.[Abstract/Free Full Text]
  46. Weber C, Springer TA. Interaction of very late antigen-4 with VCAM-1 supports transendothelial chemotaxis of monocytes by facilitating lateral migration. J. Immunol. (1998) 161:6825.[Abstract/Free Full Text]
  47. Gurtner GC, Davis V, Li H, McCoy MJ, Sharpe A, Cybulsky MI. Targeted disruption of the murine VCAM1 gene: essential role of VCAM-1 in chorioallantoic fusion and placentation. Genes. Dev. (1995) 9:1.[Abstract/Free Full Text]
  48. Collins T, Read MA, Neish AS, Whitley MZ, Thanos D, Maniatis T. Transcriptional regulation of endothelial cell adhesion molecules: NF-kappa B and cytokine-inducible enhancers. FASEB J. (1995) 9:899.[Abstract]
  49. Slakey D, Roza A, Pieper G, Johnson C, Adams M. Ascorbic acid and alpha-tocopherol prolong rat cardiac allograft survival. Transplant. Proc. (1993) 25:610.[Web of Science][Medline]
  50. Li X, Chen H, Epstein PN. Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. J. Biol. Chem. (2004) 279:765.[Abstract/Free Full Text]
  51. Wuyts WA, Vanaudenaerde BM, Dupont LJ, Van Raemdonck DE, Demedts MG, Verleden GM. N-acetylcysteine inhibits interleukin-17-induced interleukin-8 production from human airway smooth muscle cells: a possible role for anti-oxidative treatment in chronic lung rejection? J. Heart Lung Transplant. (2004) 23:122.[CrossRef][Web of Science][Medline]
  52. Gottmann U, Oltersdorf J, Schaub M, et al. Oxidative stress in chronic renal allograft nephropathy in rats: effects of long-term treatment with carvedilol, BM 91.0228, or alpha-tocopherol. J. Cardiovasc. Pharmacol. (2003) 42:442.[CrossRef][Web of Science][Medline]
  53. Cicalese L. Pyruvate in organ transplantation. JPEN J. Parenter. Enteral Nutr. (2001) 25:216.[Abstract/Free Full Text]

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