Complement activation and dysregulation of the coagulation system are pivotal issues in pig-to-human xenotransplantation. Indeed, thrombotic microangiopathy and consumptive coagulopathy appear to play a more important role than in allotransplantation, and this has increasingly been recognized as a major barrier to the success of xenotransplantation (reviewed in Satyananda et al and Schmelzle et al1,2 3-6 7-9 10 11 12,13 14 15
Activation of porcine EC by recipient antibodies and complement and/or by direct exposure to recipient platelets, immune cells, and cytokines changes the normally anticoagulant phenotype of the endothelium to a procoagulant one.16 17,18 19,20 21
The formation of thrombin on activated EC has important physiological consequences. It generates fibrin from fibrinogen22 23 14,24,25 18,26 18
Physiological hemostasis depends on a finely tuned process balancing between coagulation (clot formation) and fibrinolysis that leads to the removal of fibrin deposits (clot resolution). The key component of the fibrinolytic system is plasminogen and its active form, plasmin, which is generated by tissue plasminogen activator (tPA) and urokinase plasminogen activator.27 28 29,30
In addition to antibodies and the plasma cascade systems, human natural killer (NK) cells participate in the innate immune attack against porcine xenografts. The NK cells are able to infiltrate pig organs perfused with human blood and mediate lysis of porcine cells in vitro, either directly or via antibody-dependent cellular cytotoxicity.31-34 35,36 37-39
To study the protective effects of transgenic expression of human CD46 and HLA-E on porcine xenografts, forelimbs of wild-type and GalTKO-heterozygous, hCD46/HLA-E double transgenic pigs were ex vivo xenoperfused with whole, heparinized human blood for 12 hours as previously reported. This model represents a useful tool to study immunological, coagulatory, and fibrinolytic-antifibrinolytic responses associated with pig-to-human vascularized xenograft transplantation in the absence of hyperacute rejection. We have already shown that the transgenic expression of hCD46 provided protection against complement-mediated responses.9,40
MATERIALS AND METHODS
Ex Vivo Perfusion Model
Animal experiments in this study were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and Swiss National Guidelines. The local animal experimentation committee of the Canton Bern approved this study (permission no. BE45/11). Wild-type (n = 8) and GalTKO-heterozygous, hCD46/HLA-E double transgenic (n = 10) forelimbs of large white pigs (30-40 kg) of both sexes were used to perform ex vivo xenoperfusion with heparinized whole human blood as described previously.9,40 2 at 21% by membrane oxygenator (MEDOS Hilite 800 LT).
Tissue and Blood Sampling
Biopsies of skeletal muscle as well as serum and ethylene diamine tetra acetate (EDTA) plasma samples were obtained at baseline and after 12 hours (end) of perfusion. Snap-frozen tissue samples were used for analysis by immunofluorescence staining. Blood samples were centrifuged at 3000 rpm for 7 minutes at 4°C so separate plasma from cells, aliquoted and stored at −80°C for further analysis.
Immunofluorescence
Snap-frozen biopsy samples were cut into 5-μm-thick sections, air dried, and either processed immediately or stored at −80°C until further analysis. After fixation with acetone and hydration, the sections were stained using either 1-step direct or 2-step indirect immunofluorescence techniques. The following reagents were used: BS-I-B4 lectin (fluorescein isothiocyanate [FITC]–labeled, Sigma), goat antihuman IgM-FITC (Sigma), goat antihuman IgG-FITC (Sigma), mouse antihuman C5b-9 (Diatec), rabbit antihuman FGL2 (Aviva Systems Biology), sheep antihuman tissue factor (Affinity Biologicals), goat antihuman fibrinogen (Santa Cruz Biotechnologies), mouse antihuman PAI-1 (Hycult Biotech), rabbit antihuman tPA (Abcam). Cross-reactivity with the respective porcine antigens was verified. Secondary antibodies were goat antimouse IgG-Alexa488, donkey antigoat IgG-Alexa488, donkey antisheep IgG-Alexa488 (all from Molecular Probes), and sheep antirabbit IgG-Cy3 (Sigma). Nuclear staining was done by using 4′,6-diamidino-2-phenylindole (DAPI, Boehringer). The slides were analyzed using a fluorescence microscope (DMI4000B, Leica). Quantification of fluorescence intensity as raw integrated density was performed using Image J software, version 10.2 (National Institutes of Health) on unmanipulated TIFF images.
Markers for Thrombin Generation and Fibrinolysis
Plasma levels of hemostatic markers, including D-dimer (RayBiotech) and prothrombin fragment F1 + 2 (US Biologicals), were measured using commercially available enzyme-linked immunosorbent assay kits according to the manufacturer's instructions.
Cleavage of Human Plasminogen by Human or Porcine tPA
Cleavage of human plasminogen by human or porcine tPA was analyzed in vitro by plasmin-specific chromogenic substrate activity assay. Briefly, purified human plasminogen, as full-length protein (Abcam), was incubated at a concentration of 15 and 30 μg/mL, respectively, with either active human tPA (Abcam) or porcine tPA (Molecular Innovations) at various concentrations (0, 10, and 20 μg/mL). Plasminogen conversion to plasmin was measured as plasmin activity in the reaction mixtures using 3 mmol/L chromogenic S-2251 substrate (Chromogenix).
Inhibition of tPA-Mediated Human Plasminogen Cleavage by Porcine PAI-1
To study the inhibitory effect of porcine PAI-1 on human/porcine tPA activity, 15 and 30 μg/mL of purified human plasminogen was incubated with various concentrations of human or porcine tPA (0, 10, and 20 μg/mL) either in the presence or absence of 20 μg/mL of purified porcine active form of PAI-1 (Innovative Research) for 30 minutes at 37°C. The reaction mixtures were then used for conversion of the plasmin-specific chromogenic S-2251 substrate (3 mmol/L).
PAI-1/tPA Complex Formation
The PAI-1/tPA complexes were measured in EDTA plasma samples from ex vivo xenoperfusions by an inhouse developed Bio-Plex assay using xMAP technology, as described previously.41
Statistical Analysis
Data are shown as mean ± standard deviation. They were analyzed using GraphPad Prism 6 (GraphPad Software). Significance was tested using one-way analysis of variance with Bonferroni correction (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001).
RESULTS
Expression of the Gal Epitope and Deposition of Human IgM, IgG, and C5b-9 on Xenoperfused Tissue
The expression of the Gal epitope was analyzed by immunofluorescence on freshly frozen muscle biopsies obtained from pig forelimbs after xenoperfusion with human blood for 12 hours. Staining with the BS-I-B4 lectin showed no difference in Gal epitope expression between wild-type and GalTKO-heterozygous/hCD46/HLA-E double transgenic pig muscle tissues (Figure 1 A). Furthermore, deposition of human IgM, IgG, and C5b-9 on EC was tested at baseline and 12-hour xenoperfused tissue samples. Immunofluorescence staining showed significantly higher human IgM and IgG deposition on both wild-type (IgG: P = 0.030, IgM: P < 0.0001) and transgenic (IgG: P = 0.020, IgM: P = 0.031) xenoperfusion samples as compared to their respective baseline samples, with no significant differences between wild-type and transgenic limbs (Figure 1 B-C). Also, C5b-9 deposition was present after both wild-type (P < 0.0001) and transgenic limb (P = 0.002) xenoperfusion, but it was significantly (P < 0.0001) lower in transgenic limbs (Figure 1 D).
FIGURE 1: Expression of the Gal epitope and deposition of human IgM, IgG and C5b-9 on xenoperfused tissue. A, Gal epitope expression on wild-type and hCD4/HLA-E transgenic pig forelimb muscle samples was tested using BS-I-B4 lectin (from Bandeiraea simplicifolia ) by immunofluorescence staining. All tissue samples stained positive for Gal expression with no difference between groups. B-D, Effect of transgenic expression of hCD46 and HLA-E on immunoglobulin and complement deposition. Freshly frozen tissue samples from ex vivo xenoperfused pig limbs were analyzed for deposition of human IgM and IgG as well as C5b-9 using 1-step direct or 2-step indirect immunofluorescence staining. Representative images of (B) IgM, (C) IgG and (D) C5b-9 deposition on 12 hours perfused tissue samples. Scale bars: 75 μm. Wild-type and transgenic xenogeneic perfusion samples showed increased deposition of IgM, IgG, and C5b-9 over baseline, whereas transgenic hCD46 expression resulted in significantly reduced C5b-9 deposition. Quantitative estimation of IgM, IgG, and C5b-9 deposition was performed using Image J software, and statistical analysis was carried out by one-way ANOVA testing with Bonferroni correction. Data are mean ± SD, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; n = 8 for wild-type and n = 10 for hCD46/HLA-E transgenic pigs. ANOVA indicates analysis of variance.
In vitro, supplementary control experiments were performed using hCD46-only and hCD46/HLA-E double transgenic porcine aortic EC (PAEC). Wild-type, hCD46-only and hCD46/HLA-E transgenic PAEC were treated with normal human serum (NHS, 1:10) for 4 hours and then assessed for complement deposition as well as endothelial activation by measuring CD62E expression and complement-mediated cytotoxicity. The same cells were also grown on microcarrier beads and exposed to whole, nonanticoagulated human blood to assess clotting time. Transgenic expression of hCD46 alone or in combination with HLA-E on PAEC both resulted in significantly reduced C5b-9 deposition (P < 0.0001), CD62E expression (P < 0.0001), PAEC cytotoxicity (P < 0.0001), and prolonged clotting time (P < 0.002) as compared to wild-type PAEC (Figure S1, SDC , https://links.lww.com/TP/B158
Reduction of Endothelial Induction of FGL2 by hCD46/HLA-E
Induction of the procoagulant protein FGL2 on the vascular endothelium constitutes a direct mechanism for the production of thrombin from prothrombin. Ex vivo xenoperfusion samples showed a significantly increased FGL2 expression in wild-type (P = 0.0002) and transgenic samples (P = 0.036) as compared to their respective baseline samples. The expression of FGL2 was significantly (P = 0.028) lower in the presence of hCD46/HLA-E as compared to wild-type limb ex vivo xenoperfusions (Figure 2 A).
FIGURE 2: Reduction of endothelial expression of tissue factor, deposition of fibrin, and of plasma levels of D-dimers and prothrombin fragment F1 + 2 by hCD46/HLA-E expression on xenoperfused tissue. Xenoperfusion-induced endothelial procoagulant activity was evaluated by immunofluorescence staining on freshly frozen ex vivo xenoperfusion tissue samples for the expression of FGL2, tissue factor and deposition of fibrin as coagulation activation markers. Representative images of IF staining for (A) FGL2, (B) tissue factor and (C) fibrin on sections of muscle biopsies at baseline and after 12 hours of perfusion are shown. Expression of FGL2 and tissue factor as well as deposition of fibrin was significantly lower in samples from hCD46/HLA-E transgenic xenoperfusions as compared to wild-type xenoperfusion limbs. Immunofluorescence staining intensities of FGL2, tissue factor and fibrin were quantitatively measured using Image J software. Measurement of (D) D-dimers and (E) prothrombin fragment F1 + 2 were done in citrate plasma collected at baseline and 12 hours of xenoperfusion as soluble coagulation markers using commercial ELISA kits. After 12 hours of xenoperfusion, these markers were significantly upregulated in both wild-type and transgenic samples, but significantly lower in transgenic as compared to wild-type limb xenoperfusions. Column graphs are mean ± SD analyzed by one-way ANOVA with Bonferroni correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; n = 8 for wild-type and n = 10 for hCD46/HLA-E transgenic pigs. ELISA indicates enzyme linked immunosorbent assay; BL, baseline.
Reduction of Endothelial Expression of Tissue Factor and Deposition of Fibrin by hCD46/HLA-E
To test whether human antipig humoral responses induce a procoagulant phenotype of the pig endothelium, expression of tissue factor and deposition of fibrin were investigated by immunofluorescence staining on ex vivo xenoperfused tissue samples. Quantitative analysis using Image J software revealed a significant increase in tissue factor expression and fibrin deposition on both wild-type (tissue factor: P < 0.0001; fibrin: P = 0.0002) and transgenic (tissue factor: P = 0.004; fibrin: P = 0.003) xenoperfusion samples as compared to respective baseline samples. However, tissue factor expression (P = 0.005) and fibrin deposition (P = 0.009) were significantly reduced in transgenic xenoperfusion samples as compared to wild-type xenoperfusions (Figure 2 B-C).
Reduction of Plasma Levels of D-Dimers and Prothrombin Fragment F1 + 2 by hCD46/HLA-E
The direct measurement of thrombin is unreliable because active thrombin is rapidly neutralized by antithrombin. However, quantification of prothrombin fragment F1 + 2 allows for monitoring of the activation of the coagulation cascade, culminating in the formation of thrombin.42 P < 0.0001) and transgenic (205.4 ± 12.8 to 738.3 ± 60.5 ng/mL, P < 0.0001) limbs. However, the presence of hCD46/HLA-E transgenes resulted in significantly (P = 0.044) reduced D-dimer formation in transgenic as compared to wild-type limb xenoperfusions (Figure 2 D). Also, the prothrombin fragment F1 + 2 was significantly elevated over baseline levels after 12 hours of wild-type (27.5 ± 0.2 to 87.0 ± 8.6 ng/mL, P = 0.0001) and transgenic (27.1 ± 0.3 to 65.9 ± 14.1 ng/mL, P = 0.005) limb xenoperfusions. Comparison of wild-type versus transgenic limb xenoperfusions revealed significantly reduced prothrombin fragment F1 + 2 levels in transgenic perfusions (P = 0.031) (Figure 2 E).
Cross-Species Cleavage of Human Plasminogen by Porcine tPA and Inhibition of Human tPA by Porcine PAI-1
Tissue plasminogen activator is a key molecule in the initiation of the fibrinolytic cascade. Potentially, fibrinolysis could be compromised in pig-to-primate xenotransplantation due to a molecular incompatibility between tPA, produced by the porcine endothelium, and plasminogen in the primate blood. Therefore, we investigated the conversion of human plasminogen to plasmin by porcine tPA in vitro. As shown in Figure 3 , human plasminogen was cleaved by porcine tPA in a dose-dependent manner with no difference between human and porcine tPA activity when tested on human plasminogen. In addition, incubation of human plasminogen with either human or porcine tPA in the presence of porcine PAI-1 revealed no plasminogen conversion to plasmin. Porcine PAI-1 thus inhibits the cleavage of human plasminogen by both human and porcine tPA (Figure 3 B).
FIGURE 3: Cross-species cleavage of human plasminogen by porcine tPA and inhibition of human tPA by porcine PAI-1. A, Conversion of human plasminogen by human or porcine tPA was investigated using purified proteins in vitro and plasmin-specific chromogenic S-2251 substrate. Incubation of human plasminogen full length protein (15 and 30 μg/mL) with 0, 10 and 20 μg/mL of active human or porcine tPA protein resulted in a dose-dependent conversion of plasminogen to plasmin. No significant difference between human and porcine tPA activity was observed. B, Inhibition of human and porcine tPA activity by porcine PAI-1. Incubation of human plasminogen with human as well as porcine tPA in the presence of porcine PAI-1 showed significantly reduced plasmin activity, suggesting that porcine PAI-1 efficiently inhibited both human and porcine tPA activity on human plasminogen. Column graphs are mean ± SD analyzed by one-way ANOVA with Bonferroni correction. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, n ≥ 3 separately performed assays.
Improved Fibrinolysis by Transgenic hCD46/HLA-E Expression
Fibrinolysis was analyzed during ex vivo human-to-pig xenoperfusion by measuring tPA and PAI-1 expression. Baseline tissue samples showed strong tPA staining, mainly on vascular endothelium, which is characteristic for the healthy, profibrinolytic state of the endothelium. Activation of the endothelium is characterized by downregulation of tPA and upregulation of PAI-1 expression, leading to an antifibrinolytic endothelial phenotype. Indeed, after 12 hours of xenoperfusion, a significant decrease of tPA expression was observed in wild-type (P = 0.003 vs baseline), but not in transgenic limbs (P = 0.152 vs baseline). After 12 hours of xenoperfusion, expression of porcine tPA in wild-type pig limbs was significantly (P = 0.009) lower than that in transgenic limbs (Figure 4 A). In contrast, no PAI-1 expression was observed at baseline, neither in wild-type nor in transgenic limbs. Moreover, the expression of PAI-1 was significantly induced in both the vascular endothelium of wild-type and transgenic xenoperfused limbs (both P < 0.0001 vs baseline). The final PAI-1 expression was significantly higher in wild-type as compared to transgenic limbs (P = 0.022) at the end of the 12 hour xenoperfusion period (Figure 4 B). To determine the changes in the fibrinolytic activity also in the fluid phase, the formation of PAI-1/tPA complexes was measured in EDTA plasma samples obtained from ex vivo human-to-pig limb xenoperfusions. Baseline values of PAI-1/tPA complexes were minimal and not significantly different between wild-type and transgenic xenoperfused limbs. Formation of PAI-1/tPA complexes significantly increased after 12 hours of xenoperfusion in both wild-type (P = 0.0007 vs baseline) and transgenic limbs (P = 0.007 vs baseline). However, the presence of hCD46/HLA-E resulted in a significantly reduced PAI-1/tPA complex formation (P = 0.015; Figure 4 C).
FIGURE 4: Improved fibrinolysis by transgenic hCD46/HLA-E expression on xenoperfused tissue. A-B, Tissue samples obtained from baseline and 12 hours xenoperfused porcine limbs were stained by immunofluorescence for the expression of tPA and PAI-1. A, Expression of tPA was significantly reduced in 12 hours xenoperfused wild-type tissue samples, but not in transgenic limbs, as compared to respective baselines and xenoperfused transgenic limbs. In transgenic limbs, expression of tPA was significantly higher after 12 hours xenoperfusion than in wild-type limbs. B, PAI-1 expression was significantly upregulated after 12 hours xenoperfusion in wild-type and transgenic limbs as compared to respective baselines. However, expression of PAI-1 was significantly lower in transgenic than in wild-type limbs after 12 hours of xenoperfusion. Immunofluorescence staining intensities of tPA and PAI-1 were quantitatively measured using Image J software. C, Formation of PAI-1/tPA complexes was analyzed as fluid-phase antifibrinolytic marker in EDTA plasma samples. Significantly increased PAI-1/tPA complex formation over baseline was observed in both, transgenic and wild-type limbs after 12 hours perfusion, but the levels were significantly lower in transgenic as compared to wild-type limbs. Statistical analysis was done by one-way ANOVA testing with Bonferroni correction. Data are mean ± SD, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001; n = 8 for wild-type and n = 10 for hCD46/HLA-E transgenic pigs.
DISCUSSION
Because clot formation in the vasculature is detrimental for the affected tissue or organ, coagulation is tightly regulated on several different levels. To achieve efficient clot formation, the production of tissue factor needs to be complemented by dysfunctional or inhibited anticoagulant pathways along with suppression of fibrinolysis.43,44 45-47
Several factors contribute to hyperacute rejection of xenografts, but the key events are binding of xenoreactive natural anti-Gal antibodies to graft endothelium via Gal epitopes and the subsequent activation of the complement system.48 49,50 9 Figure S1, SDC , https://links.lww.com/TP/B158
Local expression of procoagulant proteins on the vascular endothelium is thought to play an important role in thrombus formation. FGL2, for example, is expressed and differentially regulated in many cell types, including EC.51 52,53 21,54 42 18 55 18 56
Little is known on the fibrinolytic system in xenotransplantation models. In a very early report, Jørgensen et al30 57 29 58,59
For pig-to-nonhuman primate transplantation, it is currently accepted standard to use homozygous GalTKO pigs as the “platform” on which additional genetic modifications can be assembled, such as one or more human regulators of complement activation and possibly other human proteins like thrombomodulin, TFPI, or CD39.5,60 61-63 64
In conclusion, transgenic hCD46/HLA-E expression on pig vascular endothelium resulted not only in a significant reduction of complement activation but also in prevention of the establishment of a procoagulant and antifibrinolytic state of the porcine endothelium during human-to-pig limb xenoperfusion with human blood.
ACKNOWLEDGMENTS
We thank Dr. Daniel Mettler, Mrs. Olgica Beslac and Mr. Daniel Zalokar from the Experimental Surgery Unit, as well as Alain Despont, and Julie Denoyelle, Department of Clinical Research, University of Bern, for expert technical support. Images were acquired on equipment and service supported by the Microscopy Imaging Center of the University of Bern.
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