The endothelium is an activate regulator of vascular function and inflammation, and functions as the gatekeeper regulating leukocyte entry from the blood into the tissue. Despite the importance of endothelium in regulation of vascular inflammation, and the known differences among vascular beds and tissues of origin, the intrinsic properties of specialized endothelia remain incompletely defined. Interestingly, the vessels affected by different forms of allograft rejection are distinct; in acute cellular rejection of the heart, perivascular T cell and macrophage infiltrates are found in the subendothelial space, whereas antibody-mediated rejection often manifests with intracapillary macrophages. In addition, the susceptibility of the various transplanted organs to alloimmune and ischemic injury varies widely, with the liver typically capable of withstanding substantial alloimmune insult but quite vulnerable to ischemia/reperfusion injury in contrast to heart, kidney, and lung allografts that are susceptible to cell- and antibody-mediated rejection. 

I hypothesize that the heterogeneity among organs in susceptibility to injury is shaped, at least in part, by intrinsic variation among tissue-specific endothelia. I also postulate that endothelial exposure to ischemic injury, cellular rejection, or antibody-mediated rejection stimulates the storage of inflammatory chemokines and cytokines that can be rapidly recalled upon secondary injury events inducing a spiral of heightened inflammation. This application will build upon my preliminary findings that endothelial cells exhibit rapid recall of IL-8 after exposure to transplant-relevant inflammatory stimuli. Specific questions that will be addressed in this proposal are 1) how do microvascular and large vessel endothelia from different tissues vary in their basal and inducible inflammatory states?, and 2) do EC of different origins exhibit divergent memory phenotypes after exposure to inflammatory stimuli? In order to answer these questions, I will employ an in vitro model of human endothelial cells from different vascular beds within the heart and lung to assess gene and protein expression and leukocyte recruitment capacity, and confirm protein expression in the large vessels and microvasculature in tissue sections from human hearts and lungs. In the first year of funding, I will culture and expand the endothelial cells for use in the project (approximately 6 weeks). Nanostring will be performed on unstimulated and primed EC to determine the differences in gene expression among EC types and in response to transplant-relevant stimuli. Supernatants will be analyzed for chemokine and cytokine secretion using a multiplex assay. Changes in cell surface adhesion molecule expression will be confirmed by multiparameter flow cytometry. The second year of funding will expand upon the first aim to evaluate the nature and durability of phenotype changes after inflammatory stimuli, including rapid recall of newly stored mediators. Primed cells will be challenged with a type I activator to stimulate exocytosis, and cell surface molecule expression will be measured by flow cytometry, and rapid secretion of chemokines and cytokines will be determined by multiplex assay. Confirmation of differential protein expression in human tissues will also be performed in this year.