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In xenotransplantation, the vascular endothelium serves as the first point of contact between the recipient's blood and the transplanted donor organ. The loss of the endothelium's ability to control the plasma cascades plays a critical role in the dysregulation of the complement and coagulation systems, which greatly contribute to graft rejection and hinder long-term xenograft survival. Although it is known that an intact glycocalyx is a key feature of a resting endothelium that exhibits optimal anticoagulant and anti-inflammatory properties, the role of the endothelial glycocalyx in xenotransplantation is barely investigated so far. Here, we discuss the central role of endothelial cells and the sugar-rich endothelial glycocalyx in regulating the plasma cascades, and how the loss of these functions contributes to graft damage and rejection. We highlight the importance of preserving the regulatory functions of both endothelial cells and the glycocalyx as strategies to improve xenotransplantation outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Gultom and Rieben.)

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need 1-3 . Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative 4 . Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945 days after heterotopic abdominal transplantation in baboons 5 . This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25 years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57 days and this was achieved, to our knowledge, only once 6 . Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195 days is a milestone on the way to clinical cardiac xenotransplantation 7 .

Worldwide, there is a constant rise in the number of patients with end-stage organ failure in critical need for transplants, but the number of organs/cells available from deceased or living human donors is limited. Xenotransplantation using pig organs/tissues repre-sents a potential solution for this shortage; however, it has been hampered by a number of mainly immuno-logical hurdles. Remarkable progress was presented at the latest biennial (13th) international congress of the International Xenotransplantation Association, November 2015 in Melbourne, Australia, and the American Transplant Congress, May 2016 in Boston, USA. Most importantly, the survival records of pig organ xenografts in nonhuman primate models have strikingly improved with the use of multitransgenic pigs. Moreover, no safety issues were encountered in clinical trials with porcine islets, and the removal of porcine endogenous retroviruses from the genome of a pig cell line by the CRISPR/Cas9 technology offers the perspective to overcome the perceived potential risk of xenozoonosis in the near future. For all these reasons, interest in xenotransplantation has been boosted. This review summarises the current status of xenotransplantation research, including Swiss contri-butions as well as regulatory and safety aspects in the light of upcoming clinical trials.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p < 0.0001) and significantly decreased thrombus formation compared to WT (mean thrombus coverage per viewing field in %; WT: 23.5 ± 13.0, TFkd: 2.6 ± 3.7, p < 0.0001). Our data show that a functional knockdown of TF is compatible with normal development and survival of pigs. TF knockdown could be a valuable component in the generation of multi-transgenic pigs for xenotransplantation., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Background: Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model., Methods: α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples., Results: No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions., Conclusion: Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection., (© 2014 John Wiley & Sons A/S.)

The endothelium, as an organ at the interface between the intra- and extravascular space, actively participates in maintaining an anti-inflammatory and anti-coagulant environment under physiological conditions. Severe humoral as well as cellular rejection responses, which accompany cross-species transplantation of vascularized organs as well as ischemia/reperfusion injury, primarily target the endothelium and disrupt this delicate balance. Activation of pro-inflammatory and pro-coagulant pathways often lead to irreversible injury not only of the endothelial layer but also of the entire graft, with ensuing rejection. This review focuses on strategies targeted at protecting the endothelium from such damaging effects, ranging from genetic manipulation of the donor organ to soluble, as well as membrane-targeted, protective strategies.

Background: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting., Methods: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads., Results: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS., Conclusions: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Acute vascular rejection represents a formidable barrier to clinical xenotransplantation and it is known that this type of rejection can also be initiated by xenoreactive antibodies that have limited complement-activating ability. Using a sophisticated mouse model, a recent study has provided in vivo evidence for the existence of an IgG(1)-mediated vascular rejection, which uniquely depends on both the activation of complement and interactions with FcgammaRIII on natural killer (NK) cells.

Background: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system., Method: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated., Results: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC., Conclusions: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application., (Copyright Blackwell Munksgaard, 2004)

We showed recently that low molecular weight dextran sulfate (DXS) acts as an endothelial cell (EC) protectant and prevents human complement- and NK cell-mediated 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.

Pig-to-human xenotransplantation faces the problem of hyperacute graft rejection due to the presence of human naturally occurring antibodies against the disaccharide Galalpha1-3Gal (anti-Gal antibodies) expressed on pig endothelium. Antibody-mediated complement activation is usually referred to as classical pathway activation. In this study we examined if the alternative complement pathway is also directly activated through anti-Gal antibodies or if the classical pathway is indispensable. We therefore developed a hemolysis test with rabbit erythrocytes (E), which have an activating surface for the alternative complement pathway and express abundant amounts of Galalpha1-3Gal, and used this assay in addition to the standard complement tests CH50 and AP50. In this rabbit E CH50 (RECH50) assay we were able to study activation of both major complement pathways simultaneously. FACS analysis was used to trace complement and antibody deposition on rabbit E. Anti-Gal depletion of human serum by immunoabsorption revealed a 65% reduction of rabbit E hemolysis in the RECH50 test (value before absorption: 28 +/- 5.8, after absorption: 9.9 +/- 2.8, P<0.001), but only a 35% reduction of lysis in the AP50 test (AP50 before 11.3 +/- 2.1, after 7.4 +/- 2.0, P<0.002). Repletion with purified anti-Gal fully restored hemolysis in both assays. Serum depleted of Clq showed a reduced lysis of rabbit E as compared to normal human serum; this effect increased with higher serum dilutions. The reciprocal picture, i.e. less effect on hemolysis with increasing dilution, was seen with factor D depleted serum. Comparison of the RECH50 values with the AP50 values revealed an 8.4-fold increase of lysis in the RECH50 test, in which both complement pathways are running. By FACS analysis, complement deposition on rabbit E was determined and components of the classical pathway were found, especially in sera where the alternative pathway was disrupted. We conclude that in our model anti-Gal induce lysis via both classical and alternative complement pathways, but that the alternative pathway activation is of minor importance. In addition, we saw that with higher serum dilutions, the classical pathway (i.e. anti-Gal-mediated lysis) takes a predominant role in lysing the rabbit E. As anti-Gal-mediated activation of the alternative complement cascade seems of minor importance based on our results, and as there are only few surfaces in transplanted organs that would favor the alternative pathway to be executed, the specific inhibition of early steps of the classical pathway appears as a realistic strategy in pig-to-primate xenotransplantation that-to the benefit of the patient-leaves the mainly anti-bacterial defense by the alternative pathway intact.

Dextran sulfate of 5000 molecular weight (DXS 5000) is known to block complement activation as well as the intrinsic coagulation cascade by potentiation of C inhibitor. The effect of DXS 5000 on hyperacute rejection (HAR) was tested in pig-to-human xenotransplantation models. For in vitro testing, a cytotoxicity assay was used with the pig kidney cell line PK15 as target cells and fresh, undiluted human serum as antibody and complement source. Ex vivo pig lung perfusion was chosen to assess DXS 5000 in a physiologic model. Pig lungs were perfused with fresh, citrate-anticoagulated whole human blood to which 1 or 2 mg/ml DXS 5000 were added; the lungs were ventilated and the blood de-oxygenated. Pulmonary vascular resistance (PVR) and blood oxygenation (deltapO2) were monitored throughout the experiment. Autologous pig blood and human blood without DXS 5000 served as controls. In the PK 15 assay DXS 5000 led to a complete, dose-dependent inhibition of human serum cytotoxicity with an average IC50 of 43 +/- 18 microg/ml (n=8). Pig lungs perfused with untreated human blood (n=2) underwent HAR within 105 +/- 64 min, characterized by increased PVR, decrease of deltapO2, and generalized edema. Microscopically, capillary bleeding as well as deposition of human antibodies, complement and fibrin could be observed. Addition of DXS 5000 (n=4) prolonged lung survival to 170 +/- 14 min for 1 mg/ml and 250 +/- 42 min for 2 mg/ml. and PVR values as well as edema formation were comparable to control lungs that were perfused with autologous pig blood (n=2). Activation of complement (activation products in serum, deposition on lung tissue) and the coagulation system (fibrin monomers) were significantly diminished as compared to human blood without DXS 5000. Binding of anti-Gal antibodies was not influenced, and in vitro experiments showed no evidence of complement depletion by DXS 5000. In conclusion, DXS 5000 is an efficient complement inhibitor in pig-to-human xenotransplantation models and therefore a candidate for complement-inhibitory/anti-inflammatory therapy either alone or in combination with other substances and warrants further investigation.

Pig-to-human xenotransplantation might be an option to overcome the increasing shortage of human donor organs. However, naturally occurring antibodies in human blood against the Galalpha1-->3Gal antigen on pig endothelial cells lead to hyperacute or, if prevented, acute or delayed vascular rejection of the pig graft. The purpose of this study was therefore to evaluate synthetic oligosaccharides with terminal Galalpha1-->3Gal to inhibit antigen-binding and cytotoxicity of anti-alphaGal antibodies against pig cells. Different oligosaccharides were synthesized chemically and by a combined chemico-enzymatic approach. These included monomeric di-, tri-, and pentasaccharides, a polyacrylamide-conjugate (PAA-Bdi), as well as di-, tetra-, and octamers of Galalpha1-->3Gal. All were tested for inhibitory activity by anti-alphaGal ELISA and complement-dependent cytotoxicity tests. PAA-Bdi was the best inhibitor of binding as well as cytotoxicity of anti-alphaGal antibodies. Monomeric oligosaccharides efficiently prevented binding of anti-alphaGal IgG, but less well that of anti-alphaGal IgM, with tri- and pentasaccharides showing a better efficacy than the disaccharide. The two trisaccharides Galalpha1-->3Galbeta1-->4GlcNAc and Galalpha1-->3Galbeta1-->3GlcNAc were equally effective. Oligomers of Galalpha1-->3Gal were more effective than monomers in blocking the binding of anti-alphaGal IgG. However, they could not block IgM binding, nor could they match the efficacy of PAA-Bdi. We conclude that oligosaccharides with terminal Galalpha1-->3Gal, most effectively as PAA-conjugates, can prevent binding and cytotoxicity of human anti-alphaGal in vitro. The PAA-Bdi conjugate might be most suited for use as a Sepharose-bound immunoabsorption material.

Objective: Our objective was to study lung hyperacute rejection in the pig-to-human xenotransplantation combination., Methods: Pig lungs were harvested and continuously ventilated and perfused ex vivo, using a neonatal oxygenating system, with either xenogeneic unmodified human blood (n = 6) or autogeneic pig blood (n = 6)., Results: Autoperfused lungs displayed normal hemodynamics, oxygen extraction (arteriovenous oxygen difference), and histologic characteristics throughout the 3-hour study period. By contrast, xenoperfused lungs displayed, within 30 minutes, severe pulmonary hypertension and abolishment of arteriovenous oxygen difference culminating in massive pulmonary edema, hemorrhage, and lung failure after 115 +/- 44.2 minutes of reperfusion. Within 30 minutes, the human blood showed a significant drop of anti-alpha Gal immunoglobulin M and G xenoreactive antibodies (enzyme-linked immunosorbent assay) and complement activity, consumption of clotting factors, and hemolysis; total circulating human immunoglobulins remained substantially normal. Histologically, lungs perfused with human blood were congestive and showed alveolar edema and hemorrhage and multiple fibrin and platelet thrombi obstructing the small pulmonary vessels (arterioles, capillaries, and venules) but not large (segmental or lobar) pulmonary vessels. On immunohistologic examination, deposits of human immunoglobulin M and complement (C1q and C3) proteins were observed on the alveolar capillaries., Conclusions: This pig-to-human xenograft model suggests that the pig lung perfused with human blood has an early and violent hyperacute rejection that results in irreversible pulmonary dysfunction and failure within approximately 150 minutes of reperfusion.