Gene array studies in orthotopic pig to baboon cardiac xenotransplantation

Abstract

Xenograft survival is a balance between immunological rejection and adaptive changes in the graft. Immunohistochemical analysis of vascular immunoglobulin deposition in heterotopic and orthotopic pig to primate cardiac xenografts shows vascular antibody deposition as early as 30 min after reperfusion and throughout the transplant period in both GT+ and GTKO hearts. Despite the abundance of antibody, graft contractility in heterotopic transplants and the ability of the graft to support orthotopic recipients remained high, with little evidence of microvascular thrombosis for extended periods of time. This suggests that the graft is actively adapting to chronic antibody exposure to forestall thrombosis.To investigate the effects of chronic antibody exposure we used an Affymetrix porcine gene array to study the changes in gene expression in orthotopic transplant recipients. Four sets of mRNA were studied, Group 1: normal porcine hearts were used as controls (n = 2), Group 2: long surviving pig‐to‐primate orthotopic xenografts (n = 5), Group 3: orthotopic xenografts which failed in the perioperative period to support the recipient (n = 7) and Group 4: orthotopic xenografts that were rejected (n = 3). RNA from each group was used to produce cDNA for hybridization to an Affymetrix porcine gene array (?900624). This array contains 23 935 probes. We used a previously reported extended annotation database which provides annotations for 21 122 gene titles [1]. The raw fluorescence was normalized and each probe was subjected to a present/absent test using the Affymetrix MAS 5.0 algorithm. A Student's t‐test was used to compare expression of each probe in the experimental groups (Groups 2–4) to Group 1 the control hearts. The DAVID Bioinformatics database [2] was used to identify enrichments in Kegg pathways and in Gene Ontogeny attributes.Orthotopic cardiac xenograft recipients survived for 14, 23, 34, 40 and 57 days without rejection. Recipients died due to sudden death for unknown cause, atrial hemorrhage, pulmonary hemorrhage, pneumonitis and sepsis respectively. Histologic analysis of these hearts at necropsy showed minimal injury (10%) in three recipients and moderate damage (35–45%) in two recipients. In no instance was death due to heart failure. Group 3 hearts failed within 48 hours of transplant. These hearts initially exhibited good cardiac function, based on echocardiography but could not maintain that function as they were weaned from inotropic support. Histology of the failed hearts showed vascular antibody deposition but only rarely was complement deposition (C5b) detected. There was moderate ischemic injury to the myocardium but little evidence of hemorrhage. In no instance was heart failure due to hyperacute rejection. The rejected orthotopic xenografts failed on days 9, 14 and 25. Histology showed widespread vascular antibody staining and complement deposition.Real time PCR validation of the gene array data was performed with 58 probes using RNA from the long surviving xenograft (Group 2). There was greater than 90% correlation. Groups 2–4 showed a similar number of probes which passed the exclusion test (16 006 Group 2, 16 991 Group 3 and 16 214 Group 4), however, rejected hearts (Group 4) showed the fewest number of probes with significant deviation from the control samples. Genes with highly altered levels of expression (more that 3 standard deviations from the controls) were variably regulated between Groups 2–4. For example, long surviving orthotopic hearts (Group 2) exhibited an equal proportion of genes with highly increased or decreased expression. In rejected hearts (Group 4) genes with highly altered expression were overwhelming due to decreased gene expression. All experimental groups showed increased expression of genes commonly associated with myocardial injury.Bioinformatics analysis detected enrichment in genes involved in KEGG pathways for focal adhesion, extracellular matrix receptor interactions and cell communication in the long surviving orthotopic hearts (Group 2). Additionally there was enrichment in several metabolic pathways. The changes in metabolic gene expression were also evident, to lesser degree, in the rejected hearts but not observed in hearts which failed during the perioperative period (Group 3). These results suggest that long surviving orthotopic cardiac xenografts maybe undergo extracellular remodeling and have altered metabolic activity. This analysis suggests that chronic antibody exposure may effect endothelial cell functions beyond haemostasis which promote cardiac remodeling and altered metabolism and may contribute to graft failure.This work was supported by NIH grant #U01 AI066310