Aurintricarboxylic acid inhibits endothelial activation, complement activation, and von Willebrand factor secretion in vitro and attenuates hyperacute rejection in an ex vivo model of pig-to-human pulmonary xenotransplantation

Abstract: Background: In the xenotransplantation of vascularized organs, such as the lung, a large area of endothelial cell layer is a big hurdle to be overcome. We investigated the potential protective effect of aurintricarboxylic acid (ATA), a known inhibitor of platelet adhesion, on endothelial damage induced by xenogeneic serum. We also assessed its role in hyperacute xenograft rejection using a porcine ex vivo lung perfusion model. Methods: Porcine endothelial cells were incubated with human serum and other inflammatory stimuli. For the evaluation of von Willebrand factor (vWF) secretion and tissue factor (TF) expression, we used human endothelial cells. E‐selectin expression, complement activation, TF expression and platelet activation were investigated by flow cytometry. In an ex vivo porcine lung perfusion model, the porcine lungs were perfused with fresh human whole blood: unmodified blood (n = 5), ATA‐treated blood (n = 5), and ATA and lepirudin‐treated blood (n = 5). Results: Aurintricarboxylic acid significantly inhibited TNF‐α‐ or lipopolysaccharide‐induced endothelial E‐selectin expression in a dose‐dependent manner. ATA also prevented human serum induced‐E‐selectin expression and human monocytic cell adhesion to porcine endothelial cells. Moreover, ATA abolished thrombin‐induced vWF secretion as well as complement activation. However, ATA induced endothelial TF expression and platelet activation in vitro. In ex‐vivo experiments, ATA treatment improved pulmonary function and attenuated sequestration of leukocytes. Although ATA did not influence thrombin generation, we were able to minimize its activity by adding lepirudin to the blood with ATA. Conclusions: Our study demonstrated in vitro protective effect of ATA on the inhibition of endothelial activation and vWF secretion and confirmed detrimental effect of ATA on induction of endothelial TF and platelet activation. The combination of ATA and lepirudin may act beneficially by preventing coagulation perturbation while maintaining improved xenograft survival.     

Role of anti-Gal alpha13Gal and anti-platelet antibodies in hyperacute rejection of pig lung by human blood.

BACKGROUND: Previous work has shown that antibodies against porcine antigens are an important trigger of hyperacute lung rejection (HALR). The relative importance of Gal alpha1,3Gal epitopes and other antigens, such as those expressed on pig platelet membranes or lung itself, has not been defined. This study compares the efficiency of three anti-pig antibody depletion strategies, and their efficacy with regard to attenuation of HALR. METHODS: Plasma pooled from three human donors was adsorbed against Gal alpha1,3Gal disaccharide or porcine platelet extract (PPE), or passed through pig lung vasculature. Whole blood reconstituted using adsorbed plasma was then used to perfuse piglet lung, and results were compared with unmodified human blood. RESULTS: Depletion of lung-reactive anti-Gal alpha1-3Gal antibodies was most efficient with the alphaGal column (99% +/- 0.5% vs 87% to 93% +/- 11% for PPE and 92% to 95% +/- 8% for lung, p < 0.01 vs alphaGal column). PPE column tended to be more efficient (77% to 84% +/- 12%) in removing anti-PPE antibodies than pig lung (66% to 70% +/- 14%) or the alphaGal column (56% to 63% +/- 16%, p < 0.05). Lung survival and function with each antibody depletion strategy was improved relative to unmodified controls (mean survival > or = 146 minutes vs 8 minutes for controls). Although alphaGal and lung adsorption yielded more consistent lung protection (survival beyond 2 hours) than did PPE, no approach proved significantly superior. Complement C3a elaboration at 10 minutes was attenuated > 80% by each adsorption strategy, an effect that was most pronounced in the lung adsorption group (95%, p < 0.01). Histamine elaboration was blunted significantly by PPE adsorption but not in other groups (p < 0.05). Platelet but not leukocyte sequestration was decreased with antibody depletion compared with the nondepleted group (44% to 50% vs 82%, p < 0.01). CONCLUSIONS: Each antibody depletion strategy tested significantly prolongs lung xenograft survival and function compared with unmodified human blood, but none was sufficient to reliably prevent HALR. Depletion of antibodies against both alphaGal and additional cell membrane antigens, or control of antibody-independent pathogenic pathways, may be necessary to consistently prevent HALR.     

Human complement regulatory proteins protect swine lungs from xenogeneic injury

BACKGROUND: Pulmonary xenotransplantation is not possible because of hyperacute lung injury, the pathogenesis of which is unknown. This study evaluates complement-dependent pathways of pulmonary injury during heterologous perfusion of swine lungs. METHODS: Lungs from unmodified swine and swine expressing human decay-accelerating factor and human CD59 (hDAF/hCD59 swine) were perfused with either human plasma or baboon blood. Pulmonary vascular resistance and static pulmonary compliance were measured serially, and swine lung tissue were examined by light microscopy. Complement activation was assessed by serial measurements of baboon plasma C3a-desArg concentrations. RESULTS: Perfusion of unmodified swine lungs with human plasma and baboon blood resulted in hyperacute lung injury within minutes of perfusion. However, function was preserved in swine lungs expressing human decay-accelerating factor and human CD59. In both study groups, xenogeneic perfusion with baboon blood resulted in at least a sevenfold increase in plasma C3a-desArg levels suggesting transient activation of complement. CONCLUSIONS: Lungs from swine expressing human decay-accelerating factor and human CD59 were resistant to injury during perfusion with human plasma and baboon blood, indicating that complement mediated some of the features of xenogeneic acute lung injury.     

Hyperacute lung rejection in the pig-to-human model 4: evidence for complement and antibody independent mechanisms

BACKGROUND: We assessed whether the combination of complement regulation and depletion of xenoreactive antibodies improves the outcome of pulmonary xenografts compared with either strategy alone. METHODS: Lungs from pigs heterozygous (hDAF(+/-)) or homozygous (hDAF(+/+)) for the human decay accelerating factor transgene (hDAF) or their nontransgenic litter mates (hDAF(-/-)) were perfused with heparinized whole human blood. In additional groups, xenoreactive natural antibodies (XNA) were depleted by pig lung perfusion (hDAF(-/-)/AbAbs, hDAF(+/-)/AbAbs) before the experiment. This combined approach was augmented by adding soluble complement receptor 1 (sCR1) to the perfusate in one further group (hDAF(+/-)/AbAbs/sCR1). RESULTS: HDAF(-/-) lungs perfused with unmodified human blood were rejected after 32.5 min (interquartile range, IQR 5 to 210). HDAF(+/-) lungs survived for 90 min (IQR 10 to 161, P = 0.54). Both groups showed a rapid rise in pulmonary vascular resistance (PVR), which is a characteristic feature of hyperacute rejection (HAR). This phenomenon was blunted in the hDAF(+/+) group, although survival (48 min, IQR 14 to 111) was not further prolonged. Antibody depletion (AbAbs) led to a significant increase in survival time (hDAF(-/-)/AbAbs: 315 min, IQR 230 to 427; hDAF(+/-)/AbAbs: 375 min, IQR 154 to 575), reduced PVR and less complement production. Addition of sCR1 reduced complement elaboration but did not further improve survival (200 min, IQR 128 to 580) and surprisingly tended to increase PVR. CONCLUSIONS: Depletion of xenoreactive antibodies is more effective than membrane-bound complement regulation to blunt hyperacute rejection of pulmonary xenografts, but even the combined approach including soluble-phase complement inhibition is not sufficient to reliably prevent organ failure within hours. It therefore seems likely that other factors independent of antibody and complement contribute to HAR in this model.     

Complement inhibition by FUT-175 and K76-COOH in a pig-to-human lung xenotransplant model

Abstract: Two complement inhibitors, FUT-175 (FUT) and K76-COOH (K76), were studied as single agents in an ex vivo, in situ model of pig lung rejection by human blood. Pulmonary toxicity (primarily increased pulmonary vascular resistance [PVR]) was seen with FUT at a dose which inhibited complement in vitro (0.4 mg/ml); a lower dose (0.1 mg/ml) was therefore used. K76 had little apparent toxicity at a dose which inhibited complement in vitro (6 mg/ml), but activated complement, leading to C3a elaboration. Efficacy was then assessed by 1) deposition of complement pathway components in the lung and 2) lung survival during perfusion with human blood. Neither agent consistently prolonged median lung survival (FUT: 50 min.±8 SEM; K76: 37±6), blocked thromboxane production, or prevented PVR elevation compared to experiments using unmodified human blood (survival 9 min.±2). At the doses used, both agents prevented deposition of terminal complement complex (TCC) in the lung. This finding demonstrates that the various phenomena associated with hyperacute lung rejection (thromboxane release, PVR elevation, capillary leak, and intraalveolar hemorrhage) can all occur despite abrogation of membrane attack complex formation. We can not exclude a contribution by drug toxicity or complement damage (mediated by C3a or other complement pathway components proximal to TCC) to the observed lung injury. We conclude that, although both FUT and K76 inhibit deposition of TCC in the lung, at the dose tested neither drug is useful as a single agent to prolong survival in a pig-to-human lung xenograft model.     

Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection

Nguyen B‐NH, Azimzadeh AM, Schroeder C, Buddensick T, Zhang T, Laaris A, Cochrane M, Schuurman H‐J, Sachs DH, Allan JS, Pierson RN. Absence of Gal epitope prolongs survival of swine lungs in an ex vivo model of hyperacute rejection. Xenotransplantation 2011; 18: 94–107. © 2011 John Wiley & Sons A/S. Abstract: Background: Galactosyl transferase gene knock‐out (GalTKO) swine offer a unique tool to evaluate the role of the Gal antigen in xenogenic lung hyperacute rejection. Methods: We perfused GalTKO miniature swine lungs with human blood. Results were compared with those from previous studies using wild‐type and human decay‐accelerating factor‐transgenic (hDAF^+/+) pig lungs. Results: GalTKO lungs survived 132 ± 52 min compared to 10 ± 9 min for wild‐type lungs (P = 0.001) and 45 ± 60 min for hDAF^+/+ lungs (P = 0.18). GalTKO lungs displayed stable physiologic flow and pulmonary vascular resistance (PVR) until shortly before graft demise, similar to autologous perfusion, and unlike wild‐type or hDAF^+/+ lungs. Early (15 and 60 min) complement (C3a) and platelet activation and intrapulmonary platelet deposition were significantly diminished in GalTKO lungs relative to wild‐type or hDAF^+/+ lungs. However, GalTKO lungs adsorbed cytotoxic anti‐non‐Gal antibody and elaborated high levels of thrombin; their demise was associated with increased PVR, capillary congestion, intravascular thrombi and strong CD41 deposition not seen at earlier time points. Conclusions: In summary, GalTKO lungs are substantially protected from injury but, in addition to anti‐non‐Gal antibody and complement, platelet adhesion and non‐physiologic intravascular coagulation contribute to Gal‐independent lung injury mechanisms.     

Effect of complement fragment 1 esterase inhibition on survival of human Decay-Accelerating factor pig lungs perfused with human blood

BACKGROUND: The role of complement in hyperacute lung xenograft rejection has not been fully elucidated. The present study evaluates the effect of complement (C) 1 esterase inhibition on hyperacute rejection of human decay-accelerating factor (hDAF)-positive pig lung by human blood. METHODS: Using a modification of an established ex vivo model, right and left lungs from individual animals were surgically isolated and separately perfused. Pigs homozygous for hDAF were perfused with fresh human blood that was either untreated or treated with complement 1 esterase inhibitor (C1-Inh) at doses of 1 U/ml (n = 5), 5 U/ml (n = 3) or 10 U/ml plasma (n = 5). RESULTS: Only C1-Inh at 10 U/ml prolonged survival time (230 +/- 48.3 minutes) as compared with controls (65.6 +/- 26.5 minutes, p < 0.05) and diminished complement activation (C3a and C5a, p < 0.05). Interestingly, a low concentration of C1-Inh increased the pulmonary vascular resistance (PVR; 1 U/ml: 0.54 +/- 0.3; 10 U/ml: 0.19 +/- 0.08). Sequestration of neutrophils (92 +/- 3%) and platelets (64 +/- 13%) was not prevented by any concentration of C1-Inh. Tissue deposition of C3b and C5b-9 were diminished by hDAF expression, and further blunted by treatment, with 10 U/ml C1-Inh. CONCLUSIONS: Complement plays a critical role in early events of lung hyperacute rejection (HAR). However, even potent inhibition of C1 esterase and C3/C5 convertase, using serum C1-Inh in pig lungs homozygous for hDAF expression, does not prevent rapid lung injury. Our findings implicate innate immune pathways resistant to efficient complement regulation, and suggest a role for neutrophils and platelets in the lung's particular vulnerability.     

Pre-treatment of porcine pulmonary xenograft with desmopressin: a novel strategy to attenuate platelet activation and systemic intravascular coagulation in an ex-vivo model of swine-to-human pulmonary xenotransplantation

Abstract: Background: Von Willebrand factor (vWF) has been proposed as a major contributor to the development of coagulopathy in pulmonary xenotransplantation. Pretreatment of donor swine with 1‐deamino‐8‐d ‐arginine vasopressin (DDAVP), an analog of vasopressin, can reduce the content of vWF in pulmonary xenografts. Here, we investigate the effects of DDAVP pre‐treatment in an ex‐vivo perfusion model of pulmonary xenotransplantation. Methods: We set up and performed the ex‐vivo perfusion using porcine pulmonary accessory lobes and fresh human whole blood (n = 12). Half of the donor swine were given 3 μg/kg DDAVP intravenously for 3 days before ex‐vivo perfusion (DDAVP group) and half of them were left untreated (control group). The porcine lung was perfused with fresh blood for 1 h and changes in the following parameters were monitored: pulmonary arterial pressure, pulmonary vascular resistance, blood cell counts, fibrinogen, antithrombin, platelet factor 4, D‐dimer, C3a, C4d, and xenoreactive IgM. The release of Galα1‐3Gal xenoantigen (αGal) from porcine lung which had been perfused and retained for 30 min with human blood was assessed by enzyme‐linked immunosorbent assay using αGal‐binding lectin. Results: Both DDAVP and control groups showed typical findings of immediate pulmonary dysfunction: an increase of pulmonary vascular resistance and sequestration of leukocytes and platelets after ex‐vivo perfusion. However, in the DDAVP group, the increase of platelet factor 4, C3a, and C4d after perfusion was attenuated compared to that in the control group. The release of αGal after blood retention was significantly lower in the DDAVP group than that of the control group. Conclusion: Pre‐infusion of DDAVP to the donor swine was beneficial in attenuating platelet activation as well as complement/coagulation activation. These effects of DDAVP are likely to relate to the reduction of porcine vWF content in the xenograft. Therefore, the modulation of vWF secretion in donor lungs could be an additional therapeutic way to reduce systemic coagulopathy in pulmonary xenotransplantation.     

Non-anti-Gal alpha1-3Gal antibody mechanisms are sufficient to cause hyperacute lung dysfunction in pulmonary xenotransplantation

BACKGROUND: Hyperacute lung dysfunction, which is always associated with pulmonary pig-to-primate xenotransplantation is not well understood. The mechanisms associated with its occurrence seem to differ from mechanisms involved in hyperacute xenograft rejection seen in porcine hearts or kidneys transplanted into primates. To determine the contribution of anti-Gal alpha1-3Gal antibodies (alphaGAb) in such a process, we performed a set of orthotopic pig lung transplants into baboons depleted of alphaGAb and compared graft function and survival with those receiving only immunosuppression. STUDY DESIGN: Pigs expressing human membrane cofactor protein served as donors. All baboons received triple immunosuppressive therapy. Depletion of alphaGAb in the experimental group (n = 4) was done by way of immunoadsorption using immunoaffinity membranes. Controls (n = 4) did not undergo immunoadsorption. Orthotopic lung transplants were performed through a left thoracotomy. Main pulmonary artery blood flow and pressure, left pulmonary artery blood flow, and left atrial pressure were recorded. RESULTS: At 1 hour after reperfusion, pulmonary artery graft flows and pulmonary vascular resistances (PVR) were better in animals depleted of alphaGAb than in controls (605 +/- 325.2 mL/min versus 230 +/- 21 mL/min; 27.1 +/- 41.3 mmHg/L/min versus 63 +/- 1 mmHg/L/min). But at 3 hours after reperfusion average graft flows in baboons depleted of alphaGAb had decreased to 277.6 +/- 302.2 mL/min and PVRs had increased 58.3 +/- 42.0 mmHg/L/min. On the other hand, controls maintained stable flows and PVRs (223 +/- 23 mL/min; 61 +/- 3 mmHg/L/min). Survival was ultimately better in control baboons when compared with alphaGAb depleted ones (12.2 +/- 3.3 h versus 4.4 +/- 3.2 h). CONCLUSION: Unlike heart and kidney xenograft transplants, hyperacute lung xenograft dysfunction seems to be mediated by factors other than alphaGAb.     

Hyperacute rejection of mouse lung by human blood: characterization of the model and the role of complement

BACKGROUND: The pathophysiology of hyperacute lung rejection (HALR) is not fully understood. A mouse model of HALR by human blood would be valuable to efficiently dissect the molecular mechanisms underlying this complex process, but it has not been described. METHODS: We developed a xenogenic mouse lung-perfusion model. Perfusion with heparinized autologous blood (n=3) was compared with human blood unmodified (n=7) or pretreated with C1 inhibitor (n=5) or soluble complement receptor type 1 (n=6) at unchanged flow conditions. RESULTS: Perfusion with autologous blood was associated with stable physiologic parameters and no overt evidence of lung injury for up to 2 hr. Pulmonary artery perfusion pressure increased rapidly after introduction of unmodified human blood, plasma anti-Gal(alpha)1,3Gal antibodies declined (90% immunoglobulin [Ig]M, 80% IgG), and lungs reliably met survival endpoints within 11 min (median 10 min, confidence interval [CI]: 9-11). Human Ig and neutrophils were rapidly sequestered in the lung. Survival was significantly prolonged in the soluble complement receptor type 1 group (36 min, CI: 26-46) (P<0.01) and in the C1 inhibitor group (23 min, CI: 21-25) (P<0.05), and pulmonary vascular resistance elevation and complement activation were significantly attenuated but not prevented. CONCLUSIONS: Hyperacute rejection of mouse lung by human blood occurs with kinetics, physiology, and histology closely analogous to the pig-to-human model. In addition, as in that model, neither of two potent soluble-phase complement inhibitors prevented complement activation or HALR. We conclude that the mouse lung model is relevant to dissect the cellular and molecular mechanisms governing HALR.     

Potential of an injectable polymer to prevent hyperacute rejection of ex vivo perfused porcine lungs

BACKGROUND: Removal of xenoreactive antibodies in pig-to-human lung transplantation by columns or organ perfusions proofed to be unsatisfactory and associated with adverse effects. In an ex-vivo lung perfusion model, we evaluated the potential of a soluble trisaccharide polymer (GAS914) to bind alpha-Gal antibodies and to protect a pulmonary xenograft from hyperacute rejection (HAR) and pulmonary xenograft dysfunction. METHODS: Porcine lungs were perfused with fresh human blood for 240 min. In the GAS914 treated group (n=6) the polymer was applied in three different concentrations. The control group (n=6) received no GAS914. Survival and function of perfused xenografts were monitored, and alpha-Gal antibodies as well as cytolytic anti-porcine antibodies analyzed. RESULTS: In the GAS-treated group survival of lungs was significantly prolonged, pulmonary vascular resistance reduced, pulmonary edema prevented, and oxygenation improved. On histopathological evaluation application of GAS resulted in minimal graft injury and significantly less deposition of the terminal complement complex C5b-9. Following application of GAS914, up to 89.8% of IgG alpha-Gal, 79.5% of IgM and 73.6% of anti-porcine antibodies in the human blood were bound by the polymer. Subsequent perfusion of porcine lungs resulted in absorption of only 3% of the baseline IgG alpha-Gal antibodies in the GAS914 group, compared to 87% in the controls. CONCLUSIONS: In this ex-vivo lung perfusion model, a trisaccharide polymer prevented immediate HAR, due to effective removal of alpha-Gal antibodies. In combination with additional strategies GAS914 may be a valuable tool in overcoming HAR and dysfunction of pulmonary xenografts.     

L-Arginine in lung graft preservation and reperfusion.

BACKGROUND: Inhaled nitric oxide has been shown to ameliorate early lung graft dysfunction. It improves oxygenation by inducing pulmonary vasodilatation in well-ventilated lung areas, and it also modulates leukocyte-endothelium interactions. We used a porcine, single lung transplantation model to evaluate whether the benefits of exogenously administered gas could be achieved easier by adding L-arginine, the substrate of endogenous nitric oxide synthesis, as an additive to the flush solution and intravenously during reperfusion. METHODS: Six pig lungs were flushed with modified Euro-Collins solutions containing L-arginine (2 g/liter). After cold (4 degrees C) storage, the left lung was transplanted. Ischemic time was 260 minutes. The recipients received intravenous boluses of L-arginine (30 mg/kg), followed by infusion (20 mg/kg/min) during the first 30 minutes of reperfusion. Six control animals received saline as placebo. We measured the blood flow and pulmonary vascular resistance (PVR) in the transplanted and in the native lung using a right heart bypass model. We measured blood gases, leukocyte counts, plasma free-radical trapping capacity, and diene conjugates in pulmonary venous blood and myeloperoxidase activity of the lung tissue. RESULTS: Pulmonary vascular resistance was 4 to 5-fold higher in the transplanted lung than in the native lung, which received 80% of the total blood flow. L-arginine reduced PVR by 30% in the native lung (p < 0.001), but not in the transplanted lung. L-arginine had no effect on oxygenation or carbon dioxide exchange of the transplanted lung. Nor did L-arginine treatment have any effect on leukocyte sequestration or myeloperoxidase activity in the transplanted lung. The plasma antioxidant capacity in venous blood of the transplanted lung almost doubled shortly during early reperfusion without influence of L-arginine. CONCLUSIONS: L-arginine reduced PVR in the native lung but did not improve pulmonary hemodynamics, gas exchange, or reduce leukocyte sequestration of the transplanted lung.     

Hyperacute lung rejection in the pig-to-human model. 2. Synergy between soluble and membrane complement inhibition

Azimzadeh A, Zorn GL III, Blair KSA, Zhang JP, Pfeiffer S, Harrison RA, Cozzi E, White DJG, Pierson RN III. Hyperacute lung rejection in the pig‐to‐human model. 2. Synergy between soluble and membrane complement inhibition. Xenotransplantation 2003; 10: 120–131. © Blackwell Munksgaard, 2003 Background. The role of complement in hyperacute lung xenograft rejection has not been elucidated. The present study evaluates the effect of complement (C) C3/C5 convertase inhibition on hyperacute rejection of pig lung by human blood. Methods. In an established ex‐vivo model, lungs from pigs heterozygous for human decay accelerating factor (hDAF), non‐transgenic littermate control pigs, or farm‐bred pigs were perfused with fresh human blood that was either unmodified or treated with soluble complement receptor type 1 (sCR1: TP10, 100 μg/ml). Results. Non‐transgenic lungs from littermate controls had a median survival time of 35 min (range 5 to 210; P=0.25 vs. farm‐bred piglets: median 5 min, range 5 to 10). Lungs expressing hDAF survived for a median of 90 min (range 10 to 161; P=0.5 and 0.01 vs. littermate and farm‐bred controls, respectively), with sCR1, whereas hDAF (–) lungs failed by 35 min (range 6 to 307), hDAF (+) lungs survived for 330 min (range 39 to 577) [P=0.002 vs. farm‐bred; P=0.08 vs. hDAF (–); P=0.17 vs. sCR1/hDAF (–)]. The rise in pulmonary vascular resistance (PVR) at 5 min was blunted only by hDAF (+) with sCR1 (0.26 ± 0.2 vs. 0.5 to 0.7 mmHg/ml/min for other groups). Plasma C3a and sC5b‐9 and tissue deposition of C5b‐9 were dramatically diminished using sCR1, and further decreased in association with hDAF. Histamine and thromboxane were produced rapidly in all groups. Conclusion. Complement plays an important role in lung HAR. However, even potent inhibition of C3/C5 convertase, both membrane bound in lung and by a soluble‐phase inhibitor in the blood, does not prevent activation of inflammatory responses known to be particularly injurious to the lung. Our findings implicate a role for innate immune pathways resistant to efficient complement regulation. The role of anti‐species antibody, coagulation pathway dysregulation, and additional environmental or genetic influences remain to be defined.     

Low molecular weight dextran sulfate prevents complement activation and delays hyperacute rejection in pig-to-human xenotransplantation models

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.     

Immunohistologic evaluation of mechanisms mediating hyperacute lung rejection, and the effect of treatment with K76-COOH, FUT-175, and anti-Gal column immunoadsorption

Although most investigators agree that lung dysfunction occurs rapidly in various pig-to-primate hyperacute lung rejection (HALR) models, the basic mechanisms mediating this phenomenon remain in question. Here we describe an immunohistochemical method for assessment of mechanisms driving HALR. Using an established model wherein piglet lungs are perfused ex vivo with human blood, six experimental groups (K76 COOH; FUT-175; K76 with FUT; anti-alpha-Gal column adsorption; column with FUT; and column with K76) and two control groups (unmodified human blood; autologous pig blood) were studied. Each lung was biopsied serially during perfusion, and assessed using an immunohistochemical technique, with vWF staining as an internal control to quantitate binding of human IgM, IgG, C3, C5b-9, properdin, and C1q. The effect of each treatment and subsequent lung perfusion on IgG and IgM anti-alpha-Gal titers(by ELISA) and on pig endothelial cell cytotoxicity were correlated with histologic findings. We found that [1] the classical complement activation pathway was activated, as has been shown for other pig organs in primate or human blood environments [2]; alternative complement pathway activation is also seen, which has not been described for other organs in pig-to-primate models, but only in the context of classical pathway activation; and [3] anti-Gal column absorption, pharmacologic inhibition of complement, or combination therapy each was associated with histologic evidence of partial protection, consistent with what would be predicted for each intervention. Further, immunohistologic differences correlated with physiologic outcomes [8] and with antibody assay results, and revealed that treatments used were incompletely effective. Our data suggest that more complete inhibition of antibody- and complement-driven pathways than was achieved in these experiments will be necessary to prevent the antibody and complement-mediated facets of hyperacute lung rejection. This immunohistologic technique may also help us identify additional pathogenic mechanisms important to eventual clinical application of pig-to-human lung xenografts.     

Pre-treatment of donor with 1-deamino-8-d-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient.

OBJECTIVE: Unlike cardiac or renal xenotransplants, the depletion of complement using cobra venom factor (CVF) does not improve pulmonary xenograft survival. Several cases suggest that the swine von Willebrand factor (vWF) may play a major role in presenting a different pathogenesis of pulmonary xenograft dysfunction from other organs. To evaluate the role of vWF and the complement system in mediating hyperacute vascular injury of pulmonary xenografts and elucidate pathogenesis of the injury, we performed swine-to-canine orthotropic single lung xenotransplantation after pre-treatment of 1-deamino-8-d-arginine vasopressin (DDAVP) and CVF. METHODS: We set up three groups for lung xenotransplantation: group I served as the control group; group II, recipients pre-treated with CVF; group III, donors pre-treated with DDAVP (9 mg/kg, 3 days)/recipients pre-treated with CVF (60 u/kg). Hemodynamic data, coagulation and complement system parameters, and grafted lung pathologies were examined serially for 3h after transplantation. RESULTS: DDAVP infusion reduced the vWF content in swine lung tissue in vivo (7.7+/-2.4 AU/mg vs 16.0+/-5.6 AU/mg, P < 0.0001). Infusion of CVF 24 h prior to transplantation effectively depleted the recipient's serum C3 and complement hemolytic activity below the detectable range. Regardless of the use of CVF, both groups I and II transplanted with unmodified grafts showed an immediate drop in leukocytes and platelet counts after transplantation. However, in group III, in recipients transplanted with DDAVP pre-treated swine lung, the platelet count did not decrease after transplantation (P = 0.0295). The decrease of plasma antithrombin and fibrinogen tended to be attenuated in group III. Light microscopic examination revealed extensive vascular thromboses in both capillary and larger vessels, as well as early pulmonary parenchymal damage in groups I and II, but were rarely observed in group III. CONCLUSIONS: Complement inhibition alone was not enough to alleviate intravascular thrombosis, the main pathology in pulmonary xenotransplantation. Pre-infusion of DDAVP to the donor animal was effective in preventing platelet sequestration and attenuated intravascular thrombosis. It is suggested that the strategies targeting vWF would be promising for successful pulmonary xenotransplantation.     

The Role of Antibodies and Von Willebrand Factor in Discordant Pulmonary Xenotransplantation

Pulmonary xenotransplantation is one potential solution to the paucity of donors but is currently limited by rapid failure of the graft. Unlike cardiac and renal xenotransplants, pulmonary xenografts release large quantities of swine von Willebrand factor (vWF). Swine vWF binds xenoreactive antibodies and is capable of activating primate platelets. The contribution of swine vWF to lung xenograft dysfunction is not entirely clear. To probe the role vWF plays in xenograft dysfunction, we traced the fate of xenoantibodies in vWF+ and von Willebrand factor-deficient (vWFD) swine lungs. These studies showed that the vast majority of xenoantibodies bind the vWF released from the vWF+ swine lung, and thus do not remain bound on lung endothelium. The vWF complexed to xenoantibody remained capable of aggregating primate platelets. With this information, we performed swine-to-baboon lung transplants using vWF+ and vWFD donors. Without vWF present to complex xenoantibodies, a picture of hyperacute rejection more typical of heart and kidney xenografts, with antibody deposition along the graft endothelium, interstitial hemorrhage, and edema occurred. These findings suggest that porcine vWF plays a major role in the pathogenesis of pulmonary xenograft dysfunction, and suggests promising strategies to treat lung xenograft dysfunction.     

Heme oxygenase does not contribute to control of basal vascular tone in isolated blood-perfused rat lung.

BACKGROUND: Vasoconstriction in pulmonary ischemia-reperfusion injury may involve dysfunction of the physiologic vasodilation of pulmonary arteries. Little is known of the relative importance of heme oxygenase (HO)/carbon monoxide (CO)-dependent vs nitric oxide synthase (NOS)/nitric oxide (NO)-dependent vasodilation of the pulmonary vasculature. We evaluated the significance of HO function on basal pulmonary vascular resistance (PVR) and compared it with the function of NOS. METHODS: Using an isolated blood-perfusion model, lungs of Lewis rats were assigned to 3 groups (n = 6/group). After stabilization, either an inhibitor of HO (tin-protoporphyrin-9 [SnPP-9]) or an inhibitor of NOS (NG-nitro-L-arginine methylester [L-NAME]) was added to the perfusate (50 micromol/liter and 1 mmol/liter as the final concentration, respectively). Lungs receiving saline served as controls. Gas exchange, hemodynamic and respiratory functions and the levels of cyclic 3',5'-guanosine monophosphate (cGMP) in the perfusate were measured. RESULTS: Inhibition of NOS by L-NAME resulted in a significant (p < 0.01) increase in PVR (DeltaPVR: 0.110 +/- 0.012 cm H(2)O/ml. min) within 5 minutes. In contrast, PVR was minimally affected by SnPP-9 (DeltaPVR: 0.005 +/- 0.005 cm H(2)O/ml. min), which was comparable to control lungs (DeltaPVR: 0.012 +/- 0.005 cm H(2)O/ml. min). The level of cGMP in the perfusate 5 minutes after drug application was markedly, but not significantly, lower in the L-NAME group (1.67 +/- 0.74 nmol/liter) when compared with controls (2.69 +/- 0.89 nmol/liter) and SnPP-9-treated lungs (2.65 +/- 0.66 nmol/liter). CONCLUSIONS: NOS but not HO contributes to the control of basal vascular tone in the rat lung.     

Contribution of the bronchial circulation to lung preservation

Short preservation time still severely limits lung transplantation. To determine the effect of bronchial arterial flush preservation, we studied 54 dogs using the isolated perfused working lung model. After baseline measurements, lungs were flushed with lactated Ringer's solution (60 ml/kg at 8 degrees C) by one of three methods: pulmonary artery perfusion, bronchial artery perfusion through a 15 cm closed aortic segment, or simultaneous pulmonary-bronchial artery perfusion. These groups were further subdivided and tested after 0, 4, and 17 hours of storage at 4 degrees C (n = 6 each). Lungs were ventilated (flow rate 140 ml/kg/min; inspired oxygen fraction 0.21) and continuously reperfused with normothermic deoxygenated autologous blood in a closed loop. Measured variables were hemodynamics, aerodynamics, and leukocytes in bronchoalveolar lavage. Survival time was determined from initial reperfusion to failure of the lung to oxygenate. After 0 and 4 hours of storage, there was no significant difference in survival times. After 17 hours, lungs subjected to pulmonary-bronchial artery perfusion survived longer than those perfused via either the pulmonary or bronchial arteries alone (120 +/- 24 versus 38 +/- 14 or 52 +/- 16 minutes; p less than 0.01). Pulmonary artery pressure and resistance in all groups except at failure were never different from baseline values in the intact animal. Shunts in the pulmonary-bronchial artery perfusion groups were closest to baseline at onset (8% +/- 4%) and remained lower throughout reperfusion than in the groups subjected to pulmonary or bronchial artery perfusion alone. After 17 hours, static compliance of pulmonary artery-perfused lungs was worse than baseline (1.1 +/- 0.2 x 10(-2) versus 3.2 +/- 0.7 x 10(-2) L/cm H2O/sec; p less than 0.05), whereas compliance in the pulmonary-bronchial artery perfusion groups remained constant (3.6 +/- 1.5 x 10(-2) L/cm H2O/sec). Elastic work performed by lungs subjected to pulmonary-bronchial artery flushing at onset was significantly lower when these lungs were reperfused immediately (201 +/- 14 versus 295 +/- 35 gm-m/min for pulmonary artery-flushed lungs) or after 4 hours of storage (229 +/- 30 versus 290 +/- 24 gm-m/min for pulmonary artery-flushed lungs). Bronchoalveolar lavage after 17 hours in the group subjected to pulmonary bronchial artery flushing demonstrated leukocyte counts similar to those of intact lungs (45 +/- 5 versus 29 +/- 8/mm3) and significantly less than in lungs subjected to pulmonary or bronchial artery flushing (137 +/- 18 or 82 +/- 10/mm3, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)     

Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model

Tagawa T. Protease inhibitor nafamostat mesilate attenuates complement activation and improves function of xenografts in a discordant lung perfusion model. Xenotransplantation 2011; 18: 315–319. © 2011 John Wiley & Sons A/S. Abstract: Background: Anti‐complement activity of nafamostat mesilate (FUT‐175) is strong including its variety of pharmacological effects. The effect of FUT‐175 for xenografts in an ex vivo guinea pig‐to‐rat lung perfusion model was evaluated. Methods: Heparinized Lewis rat blood was used to perfuse the lungs in three groups (n = 6 each). Group I used Lewis rat left lung for donor, Group X used guinea pig left lung for donor, and Group XF used guinea pig left lung for donor, which was perfused with Lewis rat blood with 0.2 mg/ml of FUT‐175. Complement activity causing 50% hemolysis (CH50) in the perfusion blood and pulmonary function either before or during perfusion were serially measured. Pathological assessments of the lungs were also carried out after perfusion. Results: The duration of satisfactory pulmonary function was significantly increased in Group XF. Complement activity causing 50% hemolysis in Group XF decreased more significantly compared to Group X. FUT‐175 suppressed both the increase in pulmonary arterial pressure and airway resistance, and the decrease in dynamic lung compliance. In Group X, pathology showed intra‐alveolar hemorrhage, perivascular edema, and medial thickening with endothelial swelling of the pulmonary arteries. In Group XF, less changes were observed compared to Group X. Group X showed deposition of IgM, IgG, and C3 at the endothelium of arteries, which was fewer in Group XF, and even fewer in Group I. Conclusions: This study suggests that FUT‐175 inhibited complement activation and improved lung xenograft function. FUT‐175 ameliorates hyperacute rejection in a guinea pig‐to‐rat ex vivo xenogeneic lung perfusion model.