An Approach to Controlling Inflammation and Coagulation in Pig-to-Baboon Cardiac Xenotransplantation

Abstract

Introduction: Inflammatory responses and coagulation disorders are a relevant challenge for successful cardiac xenotransplantation on its way to the clinic. To cope with this, an effective and clinically practicable anti-inflammatory and anti-coagulatory regimen is needed. The inflammatory and coagulatory response can be reduced by genetic engineering of the organ-source pigs. Furthermore, there are several therapeutic strategies to prevent or reduce inflammatory responses and coagulation disorders following xenotransplantation. However, it is still unclear, which combination of drugs should be used in the clinical setting. To elucidate this, we present data from pig-to-baboon orthotopic cardiac xenotransplantation experiments using a combination of several anti-inflammatory drugs.

Methods: Genetically modified piglets (GGTA1-KO, hCD46/hTBM transgenic) were used for orthotopic cardiac xenotransplantation into captive-bred baboons (n = 14). All animals received an anti-inflammatory drug therapy including a C1 esterase inhibitor, an IL-6 receptor antagonist, a TNF-α inhibitor, and an IL-1 receptor antagonist. As an additive medication, acetylsalicylic acid and unfractionated heparin were administered. The immunosuppressive regimen was based on CD40/CD40L co-stimulation blockade. During the experiments, leukocyte counts, levels of C-reactive protein (CRP) as well as systemic cytokine and chemokine levels and coagulation parameters were assessed at multiple timepoints. Four animals were excluded from further data analyses due to porcine cytomegalovirus/porcine roseolovirus (PCMV/PRV) infections (n = 2) or technical failures (n = 2).

Results: Leukocyte counts showed a relevant perioperative decrease, CRP levels an increase. In the postoperative period, leukocyte counts remained consistently within normal ranges, CRP levels showed three further peaks after about 35, 50, and 80 postoperative days. Analyses of cytokines and chemokines revealed different patterns. Some cytokines, like IL-8, increased about 2-fold in the perioperative period, but then decreased to levels comparable to the preoperative values or even lower. Other cytokines, such as IL-12/IL-23, decreased in the perioperative period and stayed at these levels. Besides perioperative decreases, there were no relevant alterations observed in coagulation parameters. In summary, all parameters showed an unremarkable course with regard to inflammatory responses and coagulation disorders following cardiac xenotransplantation and thus showed the effectiveness of our approach.

Conclusion: Our preclinical experience with the anti-inflammatory drug therapy proved that controlling of inflammation and coagulation disorders in xenotransplantation is possible and well-practicable under the condition that transmission of pathogens, especially of PCMV/PRV to the recipient is prevented because PCMV/PRV also induces inflammation and coagulation disorders. Our anti-inflammatory regimen should also be applicable and effective in the clinical setting of cardiac xenotransplantation.

Keywords: coagulation; heart; inflammation; orthotopic heart transplantation; xenotransplantation.

FIGURE 1 |

Application scheme of the anti-inflammatory medication.

FIGURE 2 |

Survival of the 10 animals included in the current analysis. Because 6 out of 10 experiments were deliberately terminated after 90 postoperative days (dotted line), further data analyses were limited to this period.

FIGURE 3 |

Serum leukocyte count (A) and CRP levels (B). Levels of both parameters showed distinct changes in the perioperative period but subsequently presented in normal ranges in all animals. Leukocyte counts remained within normal ranges over the whole period of 90 days. CRP levels presented with three peaks around postoperative days 35, 50, and 80 but also dropped to normal ranges after these peaks. Mean ± SD, n = 10.

FIGURE 4 |

Systemic levels of different cytokines and chemokines, plotted as fold increases compared to the preoperative levels. IL-8 (A), IL-10 (B), Eotaxin (C), MCP-1 (D), and MIF (E) increased in the perioperative period but subsequently presented in the preoperative ranges. IL-12/IL-23 (F), MDC (G), RANTES (H), and INF-γ (I) showed no relevant increase in the perioperative period and returned to their preoperative or lower ranges afterward. IL-6 (J) presented with a different course with an about 100-fold increase in the perioperative period and at least 10-fold increase in the further course of the experiments. Mean ± SEM, n = 10.

FIGURE 4 |

Systemic levels of different cytokines and chemokines, plotted as fold increases compared to the preoperative levels. IL-8 (A), IL-10 (B), Eotaxin (C), MCP-1 (D), and MIF (E) increased in the perioperative period but subsequently presented in the preoperative ranges. IL-12/IL-23 (F), MDC (G), RANTES (H), and INF-γ (I) showed no relevant increase in the perioperative period and returned to their preoperative or lower ranges afterward. IL-6 (J) presented with a different course with an about 100-fold increase in the perioperative period and at least 10-fold increase in the further course of the experiments. Mean ± SEM, n = 10.

FIGURE 5 |

Serum platelet count (A), fibrinogen levels (B), and prothrombin time (C). Levels of all parameters decreased in the perioperative period but subsequently increased to normal ranges in the first postoperative days and remained within these ranges until the end of the observation period. Mean ± SD, n = 10.