Impaired Endothelial Nitric Oxide Synthase Homodimer Formation Triggers Development of Transplant Vasculopathy - Insights from a Murine Aortic Transplantation Model

Abstract

Transplant vasculopathy (TV) represents a major obstacle to long-term graft survival and correlates with severity of ischemia reperfusion injury (IRI). Donor administration of the nitric oxide synthases (NOS) co-factor tetrahydrobiopterin has been shown to prevent IRI. Herein, we analysed whether tetrahydrobiopterin is also involved in TV development. Using a fully allogeneic mismatched (BALB/c to C57BL/6) murine aortic transplantation model grafts subjected to long cold ischemia time developed severe TV with intimal hyperplasia (α-smooth muscle actin positive cells in the neointima) and endothelial activation (increased P-selectin expression). Donor pretreatment with tetrahydrobiopterin significantly minimised these changes resulting in only marginal TV development. Severe TV observed in the non-treated group was associated with increased protein oxidation and increased occurrence of endothelial NOS monomers in the aortic grafts already during graft procurement. Tetrahydrobiopterin supplementation of the donor prevented all these early oxidative changes in the graft. Non-treated allogeneic grafts without cold ischemia time and syngeneic grafts did not develop any TV. We identified early protein oxidation and impaired endothelial NOS homodimer formation as plausible mechanistic explanation for the crucial role of IRI in triggering TV in transplanted aortic grafts. Therefore, targeting endothelial NOS in the donor represents a promising strategy to minimise TV.

Calciprotein Particles Induce Endothelial Dysfunction by Impairing Endothelial Nitric Oxide Metabolism

“…To determine the dimer/monomer protein ratio of eNOS, total protein was harvested from the cells using a Harvest Buffer (50 mmol/L Tris-HCL, 150 mmol/L NaCl and 0.5% Triton X-100). 43 Protein concentrations were measured with a protein colorimetric assay kit (#5000111, Bio-Rad). For the gel electrophoresis, samples were either boiled/denatured for 5 minutes at 95 °C followed by 10 minutes centrifugation at 14 000× g (to detect exclusively eNOS monomers) or maintained on ice (to detect intact eNOS dimers and monomers).…”

Sodium Sulfite Exacerbates Allograft Vasculopathy and Affects Tryptophan Breakdown in Murine Heterotopic Aortic Transplantation

“…The donors' thoracic aorta was retrieved and transplanted as a carotid interposition graft after 12 hours of cold ischemia time (CIT = total time on ice at 4°C). The degree of ischemia/reperfusion injury influences the severity of transplant vasculopathy [29], and 12 hours of CIT have been shown to induce slowly progressing vascular hyperplastic lesions in this major histocompatibility complex-mismatched model [30]. The preservation solution used in the present study, Custodiol® (HTK, Dr. Franz Köhler Chemie GmbH, Germany) is composed of electrolytes at concentrations similar to an intracellular concentration (sodium, calcium, potassium, and magnesium) and further contains an amino acid buffering agent, histidine/histidine hydrochloride, Trp, a-ketoglutarate, and the osmotic agent mannitol.…”

Multiple Shades of Gray—Macrophages in Acute Allograft Rejection

“…Similarly, treating the donor animal with BH4 resulted in a significant reduction in neointima formation in an aortic transplantation model. In this setting, BH4 supplementation was suggested to target eNOS preventing its uncoupling and the subsequent production of superoxide [171]. The importance of having saturated BH4 already before the major injury occurs was also observed in two ischemia preconditioning models where the increased expression of BH4 was clearly related to the protective effects of ischemic preconditioning [172,173].…”