lschemia reperfusion (I/R) injury remains a major limitation in orthotopic liver transplantation (OLT), particularly with marginal grafts. Very little is known about the mechanisms involved on leukocyte recruitment into sites of inflammatory stimulation in liver. Based upon evidence from our previous studies, we hypothesize that fibronectin (FN) is an integral part of the antigen independent cascade leading to liver I/R injury, likely providing a spatial address at which appropriate co-stimulatory signals can initiate leukocyte signaling and recruitment. We expect this proposal will unveil novel mechanisms and potential targets against I/R injury. Therefore, we propose to address the following specific aims: 1 . To analyze the function of distinct FN-alpha4beta1/alpha5beta1interactions upon leukocyte recruitment and cytokine release in liver I/R injury: Livers from genetically fat Zucker and non-steatotic SD rats will be stored at 4C in UW before being transplanted into syngeneic lean Zucker/SD rats, and the three distinct FN (EIIIA)-alpha4beta1, FN (CS1)-alpha4beta1,and FN (RGD)-alpha5beta1 interactions will be targeted with specific peptides and function-blocking mAbs. The distinct therapeutic manipulation upon (i) hepatic function and survival; (ii) leukocyte adhesion/migration (in vivo and in-vitro), and (iii) cytokine expression, will be probed. 2. To dissect mechanisms of FN-alpha4beta1/alpha5beta1 integrin action upon metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) expression in liver I/R Injury: We plan to (i) determine the contribution of distinct FN cell binding sites upon MMP-2 and MMP-9 expression/activation, and respective inhibitors (TIMP-2 and TIMP-1) in rat OLT; (ii) identify the individual sources of MMP-2 and MMP-9 in liver grafts; and (iii) evaluate the functional significance of MMP-2 and MMP-9 expression in the mechanisms of FN-integrin interactions upon liver I/R injury. 3. To explore the interplay between inducible nitric oxide synthase (iNOS), MMPs, and cytokines in the context of FN in liver I/R injury: First, the functional significance of iNOS expression will be probed in vivo with 1400W, a specific iNOS inhibitor. Second, we will dissect the interplay between TNF-alpha or other relevant cytokines, and iNOS upon FN regulation of MMP expression/activation. Third, we will unveil intracellular mechanisms, such as MAPKinase transduction pathways upon leukocyte adhesion to fibronectin and MMP expression.
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