Tetrahydrobiopterin and ischemia-reperfusion injury

Ischemia reperfusion injury remains a major quest despite several advances in organ preservation, in surgical techniques as well as in immunosuppressive therapy improving dramatically patient and graft survival. Especially in pancreas transplantation ischemia reperfusion injury associated graft pancreatitis is thought to be responsible for up to 10% of early graft losses. In previous work we have observed that treatment of animals with tetrahydrobiopterin can dramatically reduce ischemia reperfusion injury in a murine model of pancreas ischemia reperfusion injury. Tetrahydrobiopterin is a compound structurally related to the vitamins folic acid and riboflavin, but endogenously synthesized in mammals. It acts as cofactor of aromatic amino acid hydroxylases, glyceryl ether monooxygenase and nitric oxide synthases. When tetrahydrobiopterin is partially oxidized in the body to dihydrobiopterin by oxidative stress, nitric oxide synthase is known to produce oxygen radicals rather than nitric oxide by uncoupling, a phenomenon that is thought to contribute not only to ischemia reperfusion injury, but also to vascular dysfunction in a variety of diseases such as diabetes. In the present project we want to characterize the mechanisms leading to this beneficial effect of tetrahydrobiopterin. In a first approach, we want to compare the effect to other biological reducing agents and characterize which parts of the chemical structure of tetrahydrobiopterin are necessary for the observed effects. In a second approach, we want to test the influence of the three nitric oxide synthase isoforms on this effect with the help of mouse mutants lacking one of the three isoforms of nitric oxide synthase. In addition to standard clinical and biochemical parameters, we will use in vivo fluorescence microscopy to stain and quantify the microcirculation in the tissue and global protein and gene expression studies to get a comprehensive picture of the biochemical effects of treatment in dependence of the functional inactivation of a specific nitric oxide synthase isoform. We expect that the results of this project will deepen our understanding of the biochemistry of ischemia reperfusion injury and its prevention, and provide the basis for pharmacological intervention to prevent ischemia reperfusion injury in clinical applications.

Aim of this project was to describe the mode of action of tetrahydrobiopterin in preventing ischemia-reperfusion injury. We found that the neuronal isoform of nitric oxide synthase is a main inducer of ischemia reperfusion injury in the pancreas.Ischemia-reperfusion injury occurs when an organ is disconnected from the blood flow, e.g. to be transplanted to another individual. As the designation already suggests, much of the damage occurs when the organ is reconnected to the blood flow (reperfusion), e.g. after implantation of the organ. The ischemia-reperfusion injury is one of the major issues limiting the quality and the life span of transplantation organs. Therefore, the way how this damage occurs is intensively studied, and means to avoid this damage are searched for.Tetrahydrobiopterin is a compound with structural similarity to the vitamins folic acid and riboflavin. In contrast to these, however, it can be formed in our bodies. It is required to degrade the essential amino acid phenylalanine, to form the neurotransmitters dopamine and serotonin, and to make the signal molecule nitric oxide. Nitric oxide is formed by the enzyme nitric oxide synthase which occurs in three isoforms. The neuronal isoform is important for neurons and the brain. The endothelial isoform is located in the vessel wall and regulates the blood pressure. A third form plays an important role in the immune systems defense against pathogens.As a main result of our work we found that the neuronal isoform of nitric oxide synthase, not the endothelial isoform as expected, is a major player in ischemia-reperfusion injury. If this enzyme is absent, the mesh of blood capillaries in the pancreas shows almost no damage after taking out the organ, storing it, and implanting it again. The transplanted organs worked fine throughout the observation period. Nitric oxide synthase needs the vitamin-like compound tetrahydrobiopterin to function properly. If this compound is missing, the enzyme will produce highly toxic compounds derived from oxygen which damage the organ. Tetrahydrobiopterin is a labile compound which is destroyed by disconnection of the organ from the blood flow, causing the neuronal isoform of nitric oxide in the pancreas to form the toxic compounds derived from oxygen. If a single dose of tetrahydrobiopterin is applied before taking out the organ, this does not happen and the organ is not damaged. Likewise, if the enzyme is missing, this damage also does not occur.Our findings describe novel processes leading to damages in transplanted organs. We have identified a new, major metabolic player resulting in damaged organs. We hope therefore, that our research will help in the development of drugs to improve the quality and lifespan of transplanted organs in the future.