Effects of human TFPI and CD47 expression and selectin and integrin inhibition during GalTKO.hCD46 pig lung perfusion with human blood

Abstract

Background: Loss of barrier function when GalTKO.hCD46 porcine lungs are perfused with human blood is associated with coagulation pathway dysregulation, innate immune system activation, and rapid sequestration of human formed blood elements. Here, we evaluate whether genetic expression of human tissue factor pathway inhibitor (hTFPI) and human CD47 (hCD47), alone or with combined selectin and integrin adhesion pathway inhibitors, delays GalTKO.hCD46 porcine lung injury or modulates neutrophil and platelet sequestration. Methods:In a well-established paired ex vivo lung perfusion model, GalTKO.hCD46.hTFPI.hCD47 transgenic porcine lungs (hTFPI.hCD47, n = 7) were compared to GalTKO.hCD46 lungs (reference, n = 5). All lung donor pigs were treated with a thromboxane synthase inhibitor, anti-histamine, and anti-GPIb integrinblocking Fab, and were pre-treated with Desmopressin. In both genotypes, one lung of each pair was additionally treated with PSGL-1 and GMI-1271 (P-and E-selectin) and IB4 (CD11b/18 integrin) adhesion inhibitors (n = 6 hTFPI.hCD47, n = 3 reference).Results: All except for two reference lungs did not fail within 480 min when experiments were electively terminated. Selectin and integrin adhesion inhibitors moderately attenuated initial pulmonary vascular resistance (PVR) elevation in hTFPI.hCD47 lungs. Neutrophil sequestration was significantly delayed during the early time points following reperfusion and terminal platelet activation was attenuated in association with lungs expressing hTFPI.hCD47, but additional adhesion pathway inhibitors did not show further effects with either lung genotype.

Increased human complement pathway regulatory protein gene dose is associated with increased endothelial expression and prolonged survival during ex‐vivo perfusion of GTKO pig lungs with human blood

“…hCD46 phenotype was not associated with altered neutrophil kinetics (Figure 5C). Similarly, the initial rise in monocytes, shown in previous work to reflect elaboration of pig monocytes into the circuit, followed by sequestration of human monocytes, 33 was not observed in conjunction with GTKO.homoCD46.hCD55 lungs (Figure 4D). Monocyte counts were similar between lung hCD46 genotypes (Figure 5D) and other lung genotypes lacking CD55 (Figure 4D), with a transient rise in monocytes, shown in previous work to reflect elaboration of pig monocytes into the circuit followed by sequestration of human monocytes to account for the subsequent decline 33 …”

“…Monocyte counts were similar between lung hCD46 genotypes (Figure 5D) and other lung genotypes lacking CD55 (Figure 4D), with a transient rise in monocytes, shown in previous work to reflect elaboration of pig monocytes into the circuit followed by sequestration of human monocytes to account for the subsequent decline. 33 Platelet activation, as measured by CD62 expression on circulating platelets and βTG elaboration, was lower in association with GTKO.homoCD46.hCD55 genotype (p = .042 and p = .016, respectively; Figure 6A, B). Platelet activation tended to be lower with either GTKO.homoCD46 genotype, or with stronger expression of CD46…”

Bioinformatic analysis as a first step to predict the compatibility of hematopoiesis and immune system genes between humans and pigs

“…Therefore, these dissimilar genes should be paid attention to avoid coagulation disorders in human and pig xenogeneic combination settings. There are several important studies reporting the function similarities or dissimilarities of related proteins between humans and pigs, which are summarized with their sequence similarities and domain composition consistency identified in the present study [20][21][22][61][62][63][68][69][70][71][72][73][74][75][76][77][78][79][80] (Table 1).…”

Knock‐out of N‐glycolylneuraminic acid attenuates antibody‐mediated rejection in xenogenically perfused porcine lungs

“…The upstream trigger for thromboxane release and the injury exhibited by GalTKO.hCD46.Neu5GcKO lungs within 8 h of ex vivo perfusion may be attributable to incomplete control of complement activation, 46,47 vWF-28 or selectin-mediated 48 platelet adhesive interactions, 49,50 innate immune cell activation via absence of HLA-E 51 or human CD47, 52 or incompatibility between human pro-and anti-coagulant molecules and pig thromboregulatory molecules. 1,52,53 Evaluating each of these hypotheses separately may be limited by logistical constraints regarding production of informative pig phenotypes. Based on our prior reports showing that each of these pathways contributes to lung xenograft injury we presume each plays a significant role.…”