Molecular characterization of the regulation of endothelial barrier function during inflammation

A major feature of inflammation is the extravasation of cellular and macromolecular components of blood into the interstitium to attack and fend off an offending stimuli, thus initiating healing. When prolonged and/or uncontrolled, this process can begin to produce deleterious effects and inadvertently damage tissue. Such instances are observed in pathologies, such as atherosclerosis, organ transplant rejection and reperfusion injury. It is therefore vital to understand the various mechanisms that initiate and control the process of extravasation. Since the endothelium is the key component between blood and the interstitium, its regulation is largely responsible for control of extravasation. TNFα and IFNγ are cytokines that function in orchestrating signals between different cellular players involved in inflammation. In this dissertation, the effects of these cytokines on endothelial permeability were explored using in vivo and in vitro methods. I investigated two ways whereby the endothelial barrier could be controlled. Firstly, the molecular mechanisms at intercellular junctions could be regulated, thus influencing permeability via the paracellular pathway. One such mechanism involves cadherins which are transmembrane homotypic Ca²⁺-dependent cell-cell adhesion molecules implicated in the control of junctional organization and therefore, paracellular permeability. Secondly, manipulation of signaling pathways by cytokines that induce endothelial apoptotic cell death could create cell-sized gaps in the endothelial barrier. The results of this work demonstrated that combined cytokine treatments: (i) induced focal areas of cadherin-5 relocalization which corresponded to regions of macromolecular extravasation in a rat in vivo model and (ii) induced the upregulation and junctionalization of N-cadherin, concomitant with downregulation and decreased junctionalization of cadherin-5 in cultured endothelial cells. Thus, combined cytokine regulation of endothelial cadherin function was found central in both accommodating an initial microvascular permeability increase upon cytokine induction of inflammation and in limiting the extent of paracellular gap formation (via its effects on N-cadherin) under chronic inflammatory conditions. Furthermore, despite in vitro evidence of endothelial apoptosis, the combined cytokines induced most endothelial cells to express protective, anti-apoptotic molecules (e.g. A1) and therefore contributed to preserving endothelial barrier integrity. Through these experiments, specific mechanisms for regulating the endothelial barrier during inflammation have been identified and are better understood