Abstract

Inflammation is a well-coordinated response to infection, trauma, or tissue injury. The process is characterized for an alteration in permeability of the vessel wall to plasma proteins leading to leukocyte migration from blood to the extravascular compartment to neutralize and remove the noxious stimulus. During acute inflammation many mediators act on endothelial cells increasing microvascular permeability and promoting the expression of adhesion molecules that bind to leukocytes leading their diapedesis to the injured tissue. Nitric oxide (NO), produced by endothelial and inducible nitric oxide synthase (eNOS and iNOS respectively) is recognized as a key factor in inflammation regulating microvascular permeability and leukocyte adhesion. Whereas eNOS expression is constitutive, iNOS expression needs to be induced. The classic dogma in NO signaling establishes that all of its actions are mediated via soluble guanylate cyclase (sGC) and protein kinase G (PKG). In addition, S-nitrosation has emerged as an important NOdependent posttranslational modification (independent of sGC/PKG pathway) of free-thiol cysteines that alters the function of proteins, affecting processes of intracellular trafficking and phosphorylation. In vascular permeability we have demonstrated that in absence of eNOS there is minimal or no increase in permeability in response to inflammatory agonists and that the subcellular location of eNOS regulate development of hyperpermeability. In particular, in our previous grant we demonstrated that the increase in permeability in response to platelet activating factor (PAF) is independent of PKG activation. More importantly, these permeability changes correlated with S-nitrosation of p120 and VASP (both components of the adherens junctions). Interestingly changes in the location/interactions of these proteins at the adherents junction lead to destabilization of the endothelial barrier and increases in microvascular permeability. The identification of specific S-nitrosation sites on these proteins would greatly contribute to elucidate the molecular mechanisms that mediate the increase of endothelial permeability. In leukocyte adhesion, many reports have described that NO acts as a negative regulator of leukocyte adhesion to endothelial cells at extended times of treatment with pro-inflammatory agonists. This effect has been attributed to S-nitrosation of NF-B by iNOS inhibiting the expression of adhesion proteins. However, the fact that the same agonists that increase permeability by activating NO production also induce leukocyte adhesion, at the onset of the process, prompt the question about whether NO induced by eNOS activates leukocyte adhesion at the beginning of the inflammatory response. Our preliminary results suggest that this could be the case. The fact that leukocyte adhesion courses with increased phosphorylation and traffic of adhesion proteins to the endothelial cell surface suggest that Snitrosation could be involved. Based on this evidence we will test the following hypothesis: eNOS-S-nitrosation regulates microvascular permeability and leukocyte adhesion at the onset of the inflammatory response. Two general aims will test our hypothesis: 1) To test the role of S-nitrosation of p120 and VASP on microvascular permeability. 2) To test the role of S-nitrosation on leukocyte adhesion to endothelial cells at the onset of the inflammatory response. We will use an array of methods and techniques to accomplish our Research Proposal including cell culture, western blotting, siRNA, immunofluorescence microscopy and intravital microscopy. We will use EAhy926 cells and postcapillary venular endothelial cells (CVEC) to facilitate the cellular mechanistic approaches. To elucidate the significance of S-nitrosation of p120 and VASP in endothelial paracellular permeability we will do site directed mutagenesis. To elucidate the significance of S-nitrosation in leukocyte adhesion we will use eNOS siRNA and S-nitrosation inhibitors. We will use eNOS knockout mice to corroborate in vivo the significance of our in vitro data. We anticipate that our results demonstrating the significance of Snitrosation in microvascular permeability and leukocyte adhesion may serve as a basis for the development of new therapeutic strategies in the treatment of vascular diseases characterized by inflammation (for example: ischemia-reperfusion injury, stroke, cancer and atherosclerosis).