Lung transplantation is the standard treatment modality for patients suffering from a variety of end-stage pulmonary disease. This life-saving procedure is still hampered by two major problems. First, lung transplantation is confronted with donor organ shortage. The last decade has been marked by a significant increase in transplant procedures and patients on the waiting list. The disparity between a growth in demand and a short supply in donor organs results in longer waiting times for listed patients with a substantial risk of dying prior to transplantation. The majority of donors that are considered potential lung donors are declared dead based on brain-death criteria, the so called heart-beating donors (HBD) or donors after brain-death (DBD). A number of strategies have evolved in an attempt to deal with this problem of inadequate donor supply. There has been an increasing interest to use organs from circulation-arrested or so called non-heart-beating donors. These donors are also referred to as donors after cardiac death (DCD). The second obstacle in lung transplantation is primary graft dysfunction (PGD) or ischemia-reperfusion injury (IRI). PGD can cause early morbidity and mortality in 5-20% of the recipients and affects long-term outcome. PGD is the end-result of a series of harmful events that occur during three phases: the donor phase, the preservation phase and the reperfusion phase. In the current brain-dead organ donors (DBD) the pathophysiological events following brain stem herniation may contribute extensively to graft injury prior to retrieval. Both hemodynamic and inflammatory changes following neuro-endocrine disregulation may be responsible for a high risk of donor lung injury. However, the exact mechanisms are still poorly understood. In the non-heart-beating donors (DCD), circulatory arrest results in warm ischemia inside the donor before preservation. This warm ischemic interval is also a potential causal factor for the development of PGD, although it has been demonstrated that an interval of 60 minutes does not compromise graft function.Primary graft dysfunction shares key clinical and pathological features with acute lung injury (ALI). Similar endothelial, epithelial and inflammatory injurious processes promote the development of diffuse alveolar damage. The objective of PART I (chapter 1, 2 and 3) was to investigate the pathophysiological changes in the brain-dead heart-beating donor that will lead to donor graft injury and eventually primary graft dysfunction. We investigated these changes and compared them to the injury developing in the non-heart-beating donor due to warm ischemia. We used an isolated reperfusion model as surrogate for transplantation and opened a potential application of this system for ex vivo reconditioning of lungs prior to transplantation. Given the importance of epithelial barrier dysfunction in the development of primary graft dysfunction, we further investigated potential mechanisms of epithelial injury in PART II (chapter 3, 4 and 5) following an inflammatory stimulus and looked into potential treatment strategies with mesenchymal stem cells and angiopoietin-1. We used a transwell cell culture model of freshly isolated human alveolar type II cells to study epithelial permeability. The major findings of our project can be summarized as follows: In chapter 1 we developed a large animal model (pig) of donor lung injury following brain death. We found that functional assesment of these grafts during isolated reperfusion was inferior compared to lungs retrieved from a non-heart-beating donor. These findings support the use of non-heart-beating donors in clinical practice. In chapter 2 we demonstrated that lungs from HBD had an increased inflammatory upregulation compared to NHBD, both before and after reperfusion. Alveolar fluid clearance, an intrinsic protective mechanism of alveolar epithelial type II cells, was decreased upon reperfusion in lungs from HBD compared to NHBD. Alveolar fluid clearance is based on the active clearance of alveolar edema from the alveolar spaces to the interstitium and can be activated by the administration of b2-agonists. Treatment of the pulmonary grafts during isolated reperfusion with a b2-agonist, terbutaline, restored the alveolar fluid clearance, improved the functional performance during isolated reperfusion and had an anti-inflammatory response in lungs from HBD. In chapter 3 we demonstrated that ex vivo perfusion of human donor lungs declined for transplantation is technically feasible for a period of 2 hours. Graft parameters remained stable and biological function evaluated by secretion of coagulation factors was preserved. In chapter 4 we demonstrated that mesenchymal stem cells may restore eptihelial permeability of freshly isolated human alveolar type II cells in a transwell model. This beneficial effect was based on a paracrine mechanism. We used an injury model based on inflammatory stimulation of these cells as a model for primary graft dysfunction. In chapter 5 we identified angiopoietin-1 as an important paracrine secreted molecule by mesenchymal stem cells to restore the epithelial permeability of human alveolar type II cells. These findings were confirmed using siRNA techniques to eliminate the secretion of Ang1 by MSC and the use of rhAng1. In chapter 6 we investigated a potential mechanism how angiopoietin-1 might restore epithelial permeability in isolated alveolar type II cells. We found that the inflammatory injury to the cells disrupted the cytoskeletal organisation by formation of stress fibers. Administration of angiopoietin-1 restored the cytoskeletal actin and myosin molecules into their resting state. Activation of the small GTPase RhoA occured with increasing permeability and restoration of the permeability was associated with activation of Rac1/2/3. Our findings support the use of non-heart-beating donors in clinical practice and demonstrate that lungs from brain-dead donors might be of inferior quality. Our ex vivo reperfusion model can be used to assess lungs outside the donor and the recipient before transplantation and might offer the possibility to recondition grafts.An important aspect of donor lung injury is related to increased permeability of the alveolar epithelial cells. Potential treatment strategies might include the use of cell-based therapy with mesenchymal stem cells.