Fibronectin-α4β1 Integrin Interactions Regulate Metalloproteinase-9 Expression in Steatotic Liver Ischemia and Reperfusion Injury

Abstract

Ischemia/reperfusion injury is a major cause of the highly dysfunctional rate observed in marginal steatotic orthotopic liver transplantation. In this study, we document that the interactions between fibronectin, a key extracellular matrix protein, and its integrin receptor ␣4␤1, expressed on leukocytes, specifically up-regulated the expression and activation of metalloproteinase-9 (MMP-9, gelatinase B) in a wellestablished steatotic rat liver model of ex vivo ice-cold ischemia followed by isotransplantation. The presence of the active form of MMP-9 was accompanied by massive intragraft leukocyte infiltration, high levels of proinflammatory cytokines, such as interleukin-1␤ and tumor necrosis factor-␣, and impaired liver function. Interestingly, MMP-9 activity in steatotic liver grafts was, to a certain extent, independent of the expression of its natural inhibitor, the tissue inhibitor of metalloproteinases-1. Moreover, the blockade of fibronectin-␣4␤1-integrin interactions inhibited the expression/activation of MMP-9 in steatotic orthotopic liver transplantations without significantly affecting the expression of metalloproteinase-2 (MMP-2, gelatinase A). Finally, we identified T lymphocytes and monocytes/macrophages as major sources of MMP-9 in steatotic liver grafts. Hence, these findings reveal a novel aspect of the function of fibronectin-␣4␤1 integrin interactions that holds significance for the successful use of marginal steatotic livers in transplantation. Orthotopic liver transplantation (OLT) is an effective therapeutic modality for end-stage liver disease. The critical shortage of human donor livers has provided the rationale to identify methods that would allow successful utilization of marginal steatotic donor grafts, which are characterized by higher dysfunction rate compared with nonsteatotic OLTs. 1,2Ischemia/reperfusion (I/R) insult, an antigen-independent event associated with leukocyte adhesion/migration and release of cytokines and free radicals, plays a major role in post-I/R organ injury suffered by OLTs.3,4 Leukocyte recruitment to sites of inflammatory stimulation in liver, which is a venous-driven vascular bed with slow flow rates, is poorly understood and may require distinct cascade of adhesive events compared with other organs with higher flow rates. Fibronectin (FN), a large glycoprotein with a central role in cellular adhesion and migration, is likely a key extracellular matrix (ECM) protein involved in these events. 5The role of FN in leukocyte adhesion, migration, and activation has been extensively reported.6 -8 Moreover, we have previously shown that the so-called cellular FN, which is the most potent form of FN in promoting cell spreading and migration, 9,10 is virtually absent in naïve steatotic livers, and it is expressed very early on the sinusoidal endothelium during cold ischemia, before steatotic OLT and leukocyte recruitment at the graft site. 11Leukocyte transmigration across endothelial and ECM barriers is dependent on both adhesive and focal matrix degradation mechani...

Molecular Mediators of Liver Ischemia and Reperfusion Injury: A Brief Review

“…Additionally, matrix proteases facilitate the movement of leukocytes along the vasculature. Matrix metalloproteinase (MMP)-9, a gelatinase family member, is highly expressed after IRI (97). Its blockade results in impaired myeloperoxidase activation and decreased leukocyte accumulation (98).…”

Cyclooxygenase-2 Deficiency Enhances Th2 Immune Responses and Impairs Neutrophil Recruitment in Hepatic Ischemia/Reperfusion Injury

“…We have recently demonstrated that MMP-9 is expressed by leukocytes in damaged livers (33) and that MMP-9-specific inhibition impairs leukocyte migration in liver I/R injury (29). To evaluate whether COX-2 deficiency was linked to MMP-9 regulation in liver I/R injury, we performed zymography analyzes using SDS-PAGE/gelatin gels to access enzymatic activity in the liver specimens.…”

“…First-strand synthesis and PCR were performed as previously described (33). Transcripts were amplified with Platinum TaqDNA polymerase SuperMix (Invitrogen) on Gene Amp PCR system (Applied Biosystems).…”

Inducible Nitric Oxide Synthase Deficiency Impairs Matrix Metalloproteinase-9 Activity and Disrupts Leukocyte Migration in Hepatic Ischemia/Reperfusion Injury

“…Similarly to iNOS, MMP-9 is virtually absent in naive livers, and it is highly up-regulated in damaged livers after I/R injury. 15,19,23 In this study, we use iNOS deficient mice and mice treated with a specific iNOS inhibitor to test the hypothesis that iNOS expression has a regulatory function on MMP-9 activation in liver I/R injury. We demonstrate that specific iNOS inhibition markedly down-regulates MMP-9 activity, disrupts leukocyte migration, and reduces apoptosis in liver I/R injury.…”

“…15,16 Matrix metalloproteinase (MMP)-9 is one of two major gelatinases in the MMP family responsible for the turnover and degradation of several ECM proteins, including fibronectin, 17 a key ECM protein expressed very early by liver endothelial cells in response to injury, 18 including to I/R injury. 19 The expression of MMP-9 has been linked to numerous pathological conditions, such as tumor invasion, 20 inflammation, 17 arthritis, 21 cerebral I/R injury 22 liver I/R injury, 15,23 and liver transplantation. 24 In general, MMPs have a large propeptide containing cysteine, a catalytic domain with zinc at the active center, and a hemopexin-like domain.…”