One of the earliest events in the pathogenesis of intestinal ischemia/reperfusion (I/R) is microvascular dysfunction that is characterized by leukocyte/endothelial cell adhesive interactions (LECA) in postcapillary venules and impaired endothelium-dependent, NO-mediated vasodilatory responses (EDO) in upstream arteries and arterioles. Even though leukocytes do not roll along, adhere to, or emigrate across arteriolar endothelium in postischemic intestine, recent work indicates that I/R- induced LECA in postcapillary venules is causally linked to EDO dysfunction in arterioles. However, the mechanisms coupling l/R-induced LECA in postcapillary venules and postischemic EDO dysfunction in upstream arterioles are unknown. Our overall hypothesis is that l/R-induced EDO dysfunction in arterioles occurs by a mechanism that is triggered by LECA in postcapillary venules and involves the formation of signals in the interstitium elicited by the proteolytic activity of emigrated leukocytes that induce mast cell-dependent formation of angiotensin II. Subsequent activation of NAD(P)H oxidase in the vascular wall leads to the formation of reactive oxygen species which inactivates NO, thereby impairing EDO in arterioles. We will test this hypothesis by determining: 1) whether LECA in postcapillary venules play an obligatory role in the development of EDO in upstream arterioles; 2) the contribution of leukocyte-derived proteases (eg, matrix metalloproteinase-9 (MMP-9)) to EDO in arterioles; and 3) whether EDO dysfunction in arterioles is due to mast cell-derived chymase-dependent formation of All. Intravital microscopic approaches will be used to examine arteriolar EDO, venular LECA, and mast cell responses to I/R. A number of gene knockout animal models (P-selectin-/-, CD11/CD18-/-, ICAM-1-/-, MMP-9-/-, mast cell-deficient mice) will be used to further explore the mechanisms of impaired EDO arterioles isolated from postischemic intestine. Collectively, these aims address a novel mechanism to explain the profound disturbance in, arteriolar vasoregulatory function in tissues subjected to I/R. This work will identify new links between LECA in postcapillary venules, signals generated in the interstitium by emigrated leukocytes, and EDO dysfunction in arterioles. Given the importance of the endothelium in regulating vascular tone, these fundamentally important findings have enormous implications for our understanding of blood flow dysregulation in conditions characterized by I/R. ? ? ?
