Prolyl-hydroxylase inhibitor activating hypoxia-inducible transcription factors reduce levels of transplant arteriosclerosis in a murine aortic allograft model.

Abstract

The development of transplant arteriosclerosis, the hallmark feature of heart transplant rejection, is associated with a chronic immune response and also influenced by an initial injury to the graft through ischaemia and reperfusion. Hypoxia-inducible transcription factor (HIF)-1 pathway signalling has a protective effect against ischaemia-reperfusion injury and has already been demonstrated to ameliorate allograft nephropathy in previous animal studies. Therefore, the aim of this study was to investigate the effect of stabilization of hypoxia-inducible transcription factors with a prolyl-hydroxylase domain (PHD) inhibitor on transplant arteriosclerosis in an experimental aortic allograft model. MHC-class I mismatched C.B10-H2(b)/LilMcdJ donor thoracic aortas were heterotopically transplanted into the abdominal aorta of BALB/c mice. Donor animals received a single dose of the PHD inhibitor 2-(1-chloro-4-hydroxyisoquinoline-3-carboxamido) acetate (ICA) (40 mg/kg) or vehicle i.p. 4 h before transplantation. Intragraft HIF accumulation after ICA treatment was detected by immunohistochemistry before and after cold ischaemia (n = 5). Grafts were harvested 30 days after transplantation and analysed by histology (n = 7) and immunofluorescence (n = 7). In addition, intragraft mRNA expression for cytokines, adhesion molecules and growth factors was determined on Day 14 (n = 7). Donor preconditioning with ICA resulted in HIF accumulation in the aorta and induction of the HIF target genes vascular endothelial growth factor and transforming growth factor-beta. Vascular lesions were present in both experimental groups. However, there was significantly reduced intimal proliferation in preconditioned grafts when compared with vehicle controls [intimal proliferation 31.3 ± 8% (ICA) vs 55.3 ± 20% (control), P < 0.01]. In addition, experimental groups revealed a down-regulation of E-selectin (-57%) and MCP1 (-33%) expression after ICA pretreatment compared with controls, going along with decreased T-cell [1.4% CD4+ T-cell infiltration vs 8.4% (control) and 4.9% CD8+ T-cell infiltration vs 10.7% (control)], dendritic cell (0.6% dendritic cells infiltration vs 1.9% infiltration(control)] and macrophage infiltration [4.8% macrophages (ICA) vs 10.9% (control)] within vascular grafts. These data of an animal transplant model show that the pharmaceutical activation of HIF with endogenous up-regulation of protective target genes leads to adaptation of the graft to low oxygen-saturation and hereby attenuates the development of transplant arteriosclerosis and allograft injury. Pharmaceutical inhibition of PHDs appears to be a very attractive strategy for organ preservation that deserves further clinical evaluation.