C Blackwell Munksgaard 2003 Copyright
American Journal of Transplantation 2003; 4: 181–187 Blackwell Munksgaard
doi: 10.1046/j.1600-6143.2003.00306.x
Endothelial Cell Protection by Dextran Sulfate: A Novel Strategy to Prevent Acute Vascular Rejection in Xenotransplantation Thomas Laumoniera , Paul J. Mohacsia , Katja M. Matozana , Yara Banza , Andre´ Haeberlib , Elena Y. Korchaginac , Nicolai V. Bovinc , Bernard Vanhoved and Robert Riebena,∗ a Cardiology, Swiss Cardiovascular Center Bern, Switzerland b Department of Clinical Research, University Hospital, CH-3010 Bern, Switzerland c Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia d ITERT-INSERM U437, CHU Hotel Dieu, 44093 Nantes, France ∗ Corresponding author: Robert Rieben,
[email protected]
We showed recently that low molecular weight dextran sulfate (DXS) acts as an endothelial cell (EC) protectant and prevents human complement- and NK cellmediated cytotoxicity towards porcine cells in vitro. We therefore hypothesized that DXS, combined with cyclosporine A (CyA), could prevent acute vascular rejection (AVR) in the hamster-to-rat cardiac xenotransplantation model. Untreated, CyA-only, and DXS-only treated rats rejected their grafts within 4–5 days. Of the hearts grafted into rats receiving DXS in combination with CyA, 28% survived more than 30 days. Deposition of anti-hamster antibodies and complement was detected in long-term surviving grafts. Combined with the expression of hemoxygenase 1 (HO-1) on graft EC, these results indicate that accommodation had occurred. Complement activity was normal in rat sera after DXS injection, and while systemic inhibition of the coagulation cascade was observed 1 h after DXS injection, it was absent after 24 h. Moreover, using a fluorescein-labeled DXS (DXS-Fluo) injected 1 day after surgery, we observed a specific binding of DXS-Fluo to the xenograft endothelium. In conclusion, we show here that DXS + CyA induces long-term xenograft survival and we provide evidence that DXS might act as a local EC protectant also in vivo. Key words: Complement, dextran sulfate, endothelial cells, xenotransplantation Received 23 July 2003, revised and accepted for publication 5 September 2003
Introduction One possible solution to overcome the current shortage of human donor organs is xenotransplantation. Success in controlling hyperacute rejection (HAR) in pig-to-primate solid organ transplantations was achieved by using donor pigs transgenic for human complement regulatory proteins like DAF and/or CD59 (1). Recently, ‘knockout’ pigs for the major xenoantigen Gala1–3Gal have also been presented (2,3), but data on transplantation experiments with such organs are not yet available. If HAR is prevented, xenografts are still confronted with acute vascular rejection (AVR), which may represent the greatest hurdle for feasibility of pig-to-primate xenotransplantations. In this setting, xenografts elicit severe and acute vascular rejection linked to endothelial cell (EC) activation. Antibodies (Ab), associated with complement and possibly natural killer (NK) cells, as well as platelets and inflammatory cells, interact with graft EC and induce a proinflammatory and procoagulant surface due to synthesis and expression of various molecules like interleukin-1 and tissue factor (4). It has been reported that, under certain circumstances, when complement-mediated damage is inhibited and T-cell activation blocked, long-term survival of xenografts can be achieved in concordant xenotransplantation models. This state has been referred to as ‘accommodation’ and is associated with T-helper type 2 cytokine responses and upregulation of protective genes, such as bcl-2, A20 and hemoxygenase 1 (HO-1) (5). Accommodation is dependent on the expression of protective genes by the graft vasculature, and HO-1 contributes in a critical manner to this step (6,7). The initial stimulus promoting accommodation in preference to rejection is still unknown, but various studies support the fact that anti-EC Abs can initiate cytoprotective changes (8–10). These data also show that the endothelium is a key component in AVR, and recent findings confirm that this disease is caused by an active metabolic process and not by apoptosis of EC’s (11). Endothelial cells are covered with a layer of heparan sulfate proteoglycans (HSPG), which is crucial for the anticoagulant and anti-inflammatory properties of the endothelium. Moreover, HSPG regulate multiple functions such as leukocyte–endothelial interactions and extravasation (12) and are rapidly released under conditions of inflammation and tissue damage (13). We showed recently that low
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molecular weight dextran sulfate (DXS) acts as an EC protectant and prevents human complement- and NK cellmediated cytotoxicity towards porcine cells in vitro (14). DXS is a sulfated, linear or slightly branched polysaccharide (15) and is known to inhibit all three pathways of complement activation. Moreover, DXS is an inhibitor of the coagulation cascade (16). Accordingly, we asked whether AVR, as occurs in hamster-to-rat cardiac xenotransplantation, could be prevented by the use of DXS in combination with T-cell suppression by cyclosporine A (CyA).
Materials and Methods Cardiac xenotransplantation Care and use of animals in the present study were in compliance with national as well as international guidelines. Inbred, 5–7-week-old male rats from the Lewis 1 A (RT1a ) congeneic strain weighing ± 250 g and inbred adult Golden Syrian hamsters of ± 100 g were purchased from Janvier (Centre d’Elevage Janvier, Le Genest-Saint-Isle, France). Fifty heterotopic cardiac xenotransplantations (hamster to rat) were performed aseptically as previously described (17). Isoflurane was used as the anesthetic in all procedures, supplemented with oxygen through a semiclosed circuit inhalation system. After surgery, the rats received one intramuscular injection of 60 mg/kg Terramycin (Pfizer, New York, NY, USA). Graft survival was monitored by daily abdominal palpation, and rejection was defined as cessation of graft beating and confirmed by direct visualization and histological examination. The recipients were treated daily from the day of transplantation with 10 mg/kg CyA (Sandimmun Neoral, Novartis, Basel, Switzerland) dissolved in olive oil and administered orally. Low molecular weight dextran sulfate, MW 5000 (Sigma, St-Louis, MO, USA), diluted in sterile PBS (50 mg/mL) and filtered (0.2 lm, NalgeNunc, Rochester, NY, USA), was given intravenously every 2nd day from day 1 up to day 13 after transplantation (50 mg/kg at day 1 and 25 mg/kg from day 3–13). The animals were then maintained under CyA alone for 20–40 additional days or until graft rejection. Two recipients received one single i.v. injection of fluorescein-labeled DXS (DXS-Fluo, 25 mg/kg, diluted in sterile PBS) (14) one day after surgery. These animals were killed for analysis of the grafts 15–20 min after injection of DXS-Fluo.
Standard aPTT tests with rat citrate-plasma were performed to evaluate the effect of DXS-treatment on the coagulation system.
Histopathology and immunofluorescence Graft samples for histology were fixed in 4% formaldehyde solution, embedded in paraffin, cut into 3-lm slices and stained with hematoxylin-eosin for light microscopy. Grafts samples for immunohistochemical staining were embedded in Tissue Tek (OCT Compound, Torrance, CA, USA) and stored at −70 ◦ C until use. For analysis, 5-lm sections were fixed in methanol for 3–5 min, air-dried and hydrated in PBS. The sections were then stained using a two-step indirect immunofluorescence technique. Where no ratspecific antibodies were available, commercial anti-human reagents of the required specificity were tested for cross-reactivity with the respective rat antigens and used instead. The following antigens were included in the analysis: rat immunoglobulins (using biotinylated goat anti-rat IgG and IgM, Southern Biotechnology, Birmingham, AL, USA), complement (rabbit antihuman C3c, DAKO, Glostrup, Denmark), HO-1 (rabbit anti-human HO-1, Stressgene, San Diego, CA, USA), and von Willebrand Factor (vWF, rabbit anti-human vWF, DAKO). Streptavidin-FITC conjugate (Amersham, Bucks, UK), streptavidin R-phycoerythrin conjugate (Molecular Probes Europe, Leiden, the Netherlands), goat anti-rabbit IgG-FITC (Southern Biotechnology), or rabbit anti-goat IgG-FITC (Southern Biotechnology) were used to reveal bound 1st antibodies, and the slides mounted with SlowFade Light Antifade Kit (Molecular Probes).
Cellular ELISA Circulating anti-hamster antibodies in the recipient’s blood were measured by a cell-based indirect enzyme-linked immunosorbent assay (CELISA), as described earlier (18). The Syrian hamster kidney cell line HAK (CCL-15, American Type Culture Collection, Manassas, VA, USA) was used as the antigenic target. Briefly, 104 cells were seeded in DMEM (Cambrex, Vervier, Belgium) containing 10% FCS, 100 U/mL penicillin, 100 lg/mL streptomycin (Invitrogen Corporation, Carlsbad, CA, USA) in 96-well plates, and left to adhere for 24 h, then fixed for 20 min at 4 ◦ C with PBS containing 0.4% glutaraldehyde. Free sites were blocked by 2 h incubation at 37 ◦ C with PBS + 5% skimmed milk powder. The coated hamster cells were then incubated for 2 h at 4 ◦ C with rat serum samples diluted 1/20 in PBS + 0.05% Tween 20. Bound IgG and IgM molecules were detected using biotinylated goat anti-rat IgM or IgG (Southern Biotechnology) and streptavidin-alkaline phosphatase (Amersham). Alkaline phosphatase was revealed using 4-NPP substrate (Sigma) and the absorbance was measured at k = 405 nm. The relative amount of circulating Abs in the serum was expressed as OD405 nm .
Statistical analysis Statistical evaluation was performed using Student’s t-test, and results were considered significant if p-values were < 0.05.
Complement hemolytic assay (CH50) and activated partial thromboplastin time (aPTT) tests To evaluate the influence of DXS on systemic complement activity and on the coagulation cascade, blood was collected from six rats treated for 6 days with DXS + CyA (3 animals) or with PBS + CyA (3 animals). For reasons of animal welfare, these rats did not receive a hamster heart graft. Hemolytic assays (CH50) were used to determine classical complement pathway activities in rat serum samples. In brief, 0.1 mL of antibodysensitized sheep erythrocytes (108 /mL) were incubated with 0.1 mL of rat serum diluted 1 : 250 in veronal buffered saline containing Mg2+ and Ca2+ , and incubated for 60 min at 37 ◦ C in a shaking water bath. After adding 1.5 mL ice-cold PBS and centrifugation for 7 min at 1750 × g, hemolysis was determined in the supernatant by measuring the absorbance of released hemoglobin at 412 nm.
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Results DXS combined with CyA protects hamster-to-rat cardiac xenografts from acute vascular rejection Survival of xenogeneic hamster hearts in Lewis 1 A rats is shown in Figure 1 and Table 1. Consistent with previous reports using the same xenotransplantation model (19), rejection occurred rapidly within 4 days after transplantation in untreated (n = 6) as well as in CyA-only treated rats (n = 7). As compared to untreated recipients, DXS-only treated recipients (n = 5), showed a slightly prolonged survival of xenografts to 5.2 ± 0.4 days (p < 0.005). The American Journal of Transplantation 2003; 4: 181–187
Dextran Sulfate Prevents Acute Vascular Rejection
untreated (n=6) CyA-only (n=7) DXS-only (n=5) DXS + CyA (n=32)
100
Actuarial survival (%)
80
last DXS injection (d13)
60
40
20
0 0
10
20
30
40
50
60
(days 1, 4 and 50), and upon rejection, if occurring. No obvious signs of AVR were observed in DXS + CyA treated grafts removed at day 1 and day 4, respectively (data not shown). The histology of rejected grafts from this group showed a normal pattern of AVR (20) with hemorrhagic tissue, fibrin deposition and presence of polymorphonuclear cells, similar to the histology of the control group or the group treated with DXS alone or CyA alone (Figure 2D,E). Long-term surviving grafts appeared relatively normal. At day 50, there were only localized, small areas of myocardial damage, with focal fibrosis and little cellular infiltrate (Figure 2F). Withdrawal of CyA at day 35 after transplantation, as done in two cases, led to graft failure within 7 days, with a clear histology of cellular rejection (data not shown).
Days after transplantation
Figure 1: Dextran sulfate combined with daily cyclosporine A (CyA) is able to induce long-term survival of hamster heart xenografts in rat Lewis 1 A recipients. Actuarial survival of hamster-to-rat cardiac xenografts: ( ✉) untreated recipients ( ❡) treated with CyA only (—) treated with DXS only and (-) treated with DXS + CyA. In the DXS + CyA-treated rats, DXS injections were stopped 13 days after transplantation (indicated by arrow).
combination of DXS + CyA treatment significantly prolonged graft survival as compared with all other treatments (p < 0.005). Nine of 32 cardiac xenografts survived more than 30 days (28% long-term surviving grafts), 8 grafts (25%) were rejected in 21.3 ± 0.9 days, and 15 grafts (47%) were rejected in 7.6 ± 1.6 days (Table 1). In two animals, CyA was stopped 35 days after accommodation had occurred and these long-term surviving grafts were acutely rejected in 7 days after withdrawal of CyA (data not shown). Pathology of hamster cardiac xenografts A blinded histological analysis was done on all hamster heart sections. There were no significant lesions visible at 24 h after transplantation in either of the treatment groups (Figure 2A). At the time of rejection, the classical picture of perivascular edema, inflammatory cell infiltration (essentially macrophages and polymorphonuclear cells), intravascular thrombosis, interstitial hemorrhage and necrosis was observed for both the untreated (Figure 2B) and the DXSonly treated group (Figure 2C). In the group treated with DXS + CyA, grafts were examined at various time-points
Accommodation was induced in long-term surviving hamster xenografts Immunofluorescence staining for IgM, C3c, vWF, and HO-1 was done on hamster cardiac xenografts (Figure 3). Staining of na¨ıve hamster hearts was negative for IgM, C3c, HO-1, and positive for vWF (Figures 3 and 1st column). In DXS-only and CyA-only treated rats, we observed high levels of IgM and C3c deposition along the vessel walls of the grafts, whereas no staining for vWF and HO-1 was detectable (data not shown). In both long-term surviving and rejected hearts in the DXS + CyA treated group, vascular deposition of rat IgM and C3c was observed. In contrast to rejected hearts, long-term surviving xenografts showed extensive vWF staining along vessel walls and also expressed high levels of HO-1 in the vasculature, whereas rejected hearts did not. These results indicate that accommodation had occurred in long-term surviving hamster xenografts. Binding of fluorescein-labeled DXS (DXS-Fluo) to hamster cardiac xenograft vasculature To evaluate the capacity of DXS to bind to the endothelium in vivo, two transplanted recipients received a single injection of 25 mg/kg DXS-Fluo 1 day after surgery. Analysis of these hamster heart sections by fluorescence microscopy showed binding of DXS-Fluo to the vasculature. In parallel, both na¨ıve hamster hearts and the grafts of the DXS-Fluo treated rats were stained with an anti-vWF Ab to reveal endothelial integrity. Na¨ıve sections stained positive for vWF expression, whereas no vWF staining was detected in the hearts excised 1 day after transplantation, indicating early endothelial damage (Figure 4).
Table 1: Survival of hamster cardiac xenografts in rat Lewis 1 A recipients Group
Treatment
n
Graft survival (days)
Mean ± SD (days)
1 2 3 4
None CyA DXS DXS + CyA
6 7 5 32
3, 4, 4, 4, 4, 5 3, 3, 4, 4, 4, 5, 6 5, 5, 5, 5, 6 5–11 (n = 15) 20–22 (n = 8) >30 (n = 9)
4.0 ± 0.6 4.8 ± 1.1 5.2 ± 0.4 7.6 ± 1.6 21.3 ± 0.9 >30
∗ vs.
p p > 0.05∗ , n.s p < 0.005∗ p < 0.005∗∗
group 1. ∗∗ vs. groups 1, 2, 3.
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A
B
C
D
E
F
Figure 2: Histology of hamster cardiac xenografts. Hamster hearts were stained with hematoxylin and eosin. (A) Graft excised at day 1 after transplantation, showing nearly normal histology and minimal cellular infiltration. (B) Hamster heart rejected by an untreated rat at day 4 or (C) by a DXS-only-treated recipient at day 5, showing edema, thrombosis, myocardial necrosis and polymorphonuclear cell infiltration. (D) Graft rejected by DXS + CyA-treated recipient at day 8 and (E) at day 21. (F) Accommodated hamster heart taken at day 50 after transplantation from the DXS + CyA-treated group, showing a normal blood vessel with only slight leukocyte infiltration. The black bars represent 100 lm. The shown sections are representative of four analyzed grafts from each group.
naÏve
rejection d 4
long-term surviving
IgM
C3c
vWF
HO-1
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Figure 3: Markers of endothelial attack as well as integrity in na¨ıve, rejected, and long-term surviving hamster hearts. Hamster heart sections were tested by examining tissue for deposition of anti-donor antibodies (IgM), complement (C3c) and for the expression of hemoxygenase-1 (HO-1), an antiapoptotic gene. Moreover, endothelial integrity is demonstrated by the presence of von Willebrand Factor (vWF). The results shown are representative of 4 transplants studied in each group, and suggest that accommodation had occurred in long-term surviving hamster cardiac xenografts.
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Dextran Sulfate Prevents Acute Vascular Rejection DXS-Fluo
500
A
CH50 test
CH50 values
400 300 200 100
DXS-Fluo + CyA, day 1 vWF
0 Before injection
24hrs after injection
1hr after injection
PBS DXS
B
aPTT test Time in seconds
300
vWF
200
100
0 Before injection
naÏve hamster heart
Figure 4: Binding of fluorescein labeled DXS (DXS-Fluo) to hamster cardiac xenograft vasculature. Hamster hearts were transplanted into rats, which received a single i.v. injection of DXS-Fluo (25 mg/kg) 1 day after surgery. Fifteen minutes after injection the rats were sacrificed. Hamster heart sections were stained in parallel for vWF to evaluate endothelial integrity. The results shown are representative of 2 transplants studied.
1hr after injection
24hrs after injection
Figure 5: Effect of intravenous administration of DXS on complement and coagulation. Lewis 1 A rats were treated daily with CyA and received intravenous injections of either PBS or DXS every 2nd day for 6 days. Blood was collected before, 1 h and 24 h after the injections. Hemolytic assays for the classical pathway of complement activation (CH50 test, Figure 5A) and standard aPTT tests (Figure 5B) are shown. White columns represent the control group with PBS and black columns the DXS-treatment group. Shown are average values of the three determinations (days 1, 3, 5), with indication of the standard deviations. 2.0
*
IgM IgG
Measurement of anti-hamster antibodies Anti-hamster Ab levels measured in rat serum are shown in Figure 6. In agreement with previous data (18,21), our na¨ıve rats had very low titers of anti-hamster Ab. However, anti-hamster IgM Ab were rapidly produced in graft recipients, and significantly increased levels were measured in all treatment groups as early as 4 days after transplantation (p < 0.05, all groups compared to na¨ıve rats). CyA or DXS, alone or in combination, did not significantly influence IgM Ab titers as compared with untreated rats. In contrast to IgM, no significant increase of anti-hamster IgG could be observed in the graft recipients, with the exception of the two CyA + DXS-treated rats in which CyA was stopped American Journal of Transplantation 2003; 4: 181–187
OD (405 nm)
1.5
1.0
**
0.5
DXS – CyA
DXS + CyA
No rejection, >30d
DXS + CyA
Rejection d20-d22
DXS + CyA
Rejection d5-d11
DXS alone
Rejection d5-d6
CyA alone
Untreated
vs. DXS + CyA
Rejection d7 after CyA-stop
** P < 0.005
Rejection d3-d6
* P < 0.05
Rejection d4
0.0
Naïve
DXS does not induce systemic complement depletion and partially inhibits the coagulation cascade Standard CH50 (Figure 5A) and aPTT (Figure 5B) tests were done on rat blood to investigate the influence of DXS in vivo. Repeated administrations of either PBS + CyA or DXS + CyA did not influence the systemic complement activity. In the aPTT test we showed that DXS injection significantly increased the coagulation time to more than 300 s one hour after injection (p < 0.005), followed by recovery to normal levels within 24 h after DXS injection.
Figure 6: Measurement of anti-hamster Ab levels in serum of Lewis 1 A rats by cellular ELISA. As compared with na¨ıve animals, all treatment groups of xenograft recipients showed a significant increase (p < 0.05) in anti-hamster IgM levels (black columns), whereas IgG (white columns) was only increased in the group with CyA withdrawal at day 35. This group also showed a significant increase in anti-hamster IgM as compared with the other CyA + DXS groups. The relative amount of circulating Ab in the serum is expressed as OD405 nm . Values shown are the mean ± SD of all animals in the respective groups.
at day 35 post transplantation (p < 0.005 vs. CyA + DXS group). The withdrawal of CyA also led to an increase in anti-hamster IgM in those two rats (p < 0.05 vs. CyA + DXS group). 185
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Discussion Several strategies, in particular the use of transgenic pigs for human complement regulatory proteins (1), have been successful in preventing HAR in pig-to-primate xenotransplantation. However, to date, the majority of porcine xenografts still succumbs to AVR, and it remains unclear if the availability of knock-out pigs for the alpha1,3-galactosyltransferase (3) will solve this problem. AVR may therefore continue to be a major obstacle to the success of pig-to-human xenotransplantation. There is now clear evidence that the earliest signs of organ injury, namely proinflammatory and procoagulant changes at the surface of EC, are caused by activation of the endothelium (11). Immunomodulatory intervention that would target EC, in a way to maintain and/or restore endothelial integrity, could therefore represent a promising way to prevent xenorejection. In the present study, we examined the capacity of DXS to act as an EC protectant in vivo, in particular to prevent hamster cardiac xenografts from undergoing AVR. We demonstrated that DXS in combination with CyA significantly prolonged xenograft survival. Moreover, long-term graft survival was achieved in 9 of 32 animals (28%) treated with DXS + CyA. These xenografts showed excellent preservation of morphology. In addition, we observed expression of the protective gene HO-1 in the graft vasculature, concurrently with deposition of anti-hamster IgM as well as complement. These data are in accordance with the situation referred to as accommodation (7). Other treatments to induce accommodation in the hamster-to-rat model were reported to have success rates between 60% (19) and 100% (22), compared to which our approximately 30% accommodation rate may seem low. However, in contrast to most other protocols we did not pretreat the recipients before transplantation, but injected DXS only 24 h after the operation. We do not yet know whether this time-point is optimal for protection of EC, and it is likely that a refinement of our experimental protocol will lead to an improvement of the accommodation rate. Of the 23 non-accommodated grafts in DXS + CyAtreated recipients, 15 were rejected in 7.6 ± 1.6 and 8 in 21.3 ± 0.9 days. The histology of these grafts was similar to the pattern of AVR observed around day 4 in the untreated group, showing a picture of acute humoral xenograft rejection. In contrast to the pathology of xenograft rejection described by others for the pig to baboon model (23), no clear signs of an increased acute cellular xenograft rejection were noticed in the grafts rejected around day 21 as compared with the ones rejected at day 4 or day 7–8. It is not clear why we were able to achieve long-term surviving grafts in our model. Repeated DXS injections did not induce systemic complement depletion, nor did they affect serum levels of anti-hamster Ab. However, we recently demonstrated that DXS acts as an EC protectant and 186
prevents human complement- and NK cell-mediated cytotoxicity towards porcine cells in vitro (14). Here, we show binding of DXS-Fluo to the graft EC one day after cardiac xenotransplantation, supporting our hypothesis that DXS acts locally and might functionally replace HSPG that are known to be shed from the EC surface upon activation (13). HSPG modulate the actions of a large number of extracellular ligands (24) and are involved in the preservation of the critical anticoagulant surface of vascular EC (25). These dynamic multifunctional cell regulators participate actively in the pathophysiology of AVR. Earlier studies have tried the use of synthetic sulfated oligosaccharides to prolong the survival of cardiac xenografts by inhibiting release of HSPG from EC (26). In our study, we hypothesized that DXS could act as a ‘repair coat’ by re-establishing an anticoagulant and anti-inflammatory surface. In fact, we observed that besides prevention of local, but not systemic, complement activation, DXS had an important effect on the coagulation cascade. This effect of DXS was already described earlier (16) and might participate in our model in the regulation of coagulation disorders, which are known to play an important part in the early phase of AVR. Systemic inhibition of the coagulation cascade can limit the use of DXS in a clinical setting (27), but in our model, injecting DXS every 2nd day up to day 13 after transplantation did not cause major bleeding complications. In addition, no signs of acute toxicity of i.v. administered DXS were observed. Another hypothesis to explain the achievement of longterm surviving xenografts in this model could be associated with the capacity of the graft to accommodate. It was previously described that the initial stimulus promoting xenograft accommodation may be anti-graft Ab themselves. It was shown in vitro, that porcine EC pretreated with human anti-pig IgM became resistant over the next day to complement-mediated damage (28), and in the hamster-to-rat model Soares and coworkers hypothesized that the gradual exposure of the graft to increasing amount of anti-donor Ab could induce a protective phenotype of EC (29). We reported earlier that DXS does not inhibit human IgM deposition on porcine EC (14). Here, we show that DXS adhered to the activated xenograft endothelium and protected it from complement-mediated damage, but did not suppress Ab deposition and up-regulation of protective genes such as HO-1. In addition, we know that at least in vitro DXS does not induce up-regulation of HO-1 by itself (unpublished data). We provide evidence here that DXS is effective in preventing AVR in hamster-to-rat cardiac xenotransplantation. The exact mechanism by which DXS exerts its protective effect is not yet known, but these results support our hypothesis that DXS may act as a ‘repair coat’ and could be considered a prototype of an EC protectant, able to locally prevent tissue damage mediated by mechanisms of innate immunity, and thus allowing for up-regulation of protective genes. American Journal of Transplantation 2003; 4: 181–187
Dextran Sulfate Prevents Acute Vascular Rejection
Acknowledgments The authors wish to thank Mr Hans Liechti, University of Bern, for animal husbandry and Claire Usal, INSERM U437 for animal surgery assistance. The support of Prof. T. Schaffner, Institute of Pathology, University of Bern, Switzerland, is gratefully acknowledged. This work was supported by the Swiss Heart Foundation, the Swiss National Science Foundation (NRP 46 project no. 4046–058668 and SCOPES project no. 7SUPJ062207), and the Katharina Huber-Steiner Foundation, Bern, Switzerland.
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