The immune response to an organ allograft involves perpetuation of T cell infiltration and activation. Advances in understanding the mechanisms of T cell activation have placed particular emphasis on the interactions between the T-cell receptor and antigen presenting cells, with little reference to the fact that in vivo activation occurs in the physical context of extracellular matrix proteins (ECM). Indeed, the possibility that ECM proteins may have a determining role in lymphocyte adhesion and tissue localization and function is now becoming more appreciated in view of growing evidence indicating that integrins and other T cell antigens bind ECM components, with some of these components exerting synergistic effects on T-cell activation. This review focuses on the importance of interactions between lymphocytes and fibronectin, a prominent ECM component, for cell migration and function in organ allograft recipients. It explores novel therapeutic approaches based on the assumption that fibronectin represents an active element in the process of T cell activation in the immune cascade triggered by organ transplantation.

The liver is a large highly vascularized organ with a central function in metabolic homeostasis, detoxification, and immunity. Due to its roles, the liver is frequently exposed to various insults which can cause cell death and hepatic dysfunction. Alternatively, the liver has a remarkable ability to self-repair and regenerate after injury. Liver injury and regeneration have both been linked to complex extracellular matrix (ECM) related pathways. While normal degradation of ECM components is an important feature of tissue repair and remodeling, irregular ECM turnover contributes to a variety of liver diseases. Matrix metalloproteinases (MMPs) are the main enzymes implicated in ECM degradation. MMPs not only remodel the ECM, but also regulate immune responses. In this review, we highlight some of the MMP-attributed roles in acute and chronic liver injury and emphasize the need for further experimentation to better understand their functions during hepatic physiological conditions and disease progression.

Hepatic ischemia and reperfusion injury (IRI) is an inflammatory condition and a significant cause of morbidity and mortality after surgery. Matrix metalloproteinases (MMPs) have been widely implicated in the pathogenesis of inflammatory diseases. Among the different MMPs, gelatinases (MMP-2 and MMP-9) are within the most prominent MMPs detected during liver IRI. While the role of MMP-9 in liver damage has been fairly documented, direct evidence of the role for MMP-2 activity in hepatic IRI remains to be established. Due to the lack of suitable inhibitors to target individual MMPs in vivo, gene manipulation is as an essential tool to assess MMP direct contribution to liver injury. Hence, we used MMP-2-/- deficient mice and MMP-2+/+ wild-type littermates to examine the function of MMP-2 activity in hepatic IRI. MMP-2 expression was detected along the sinusoids of wild-type livers before and after surgery and in a small population of leukocytes post-IRI. Compared to MMP-2+/+ mice, MMP-2 null (MMP-2-/-) mice showed exacerbated liver damage at 6, 24, and 48 hours post-reperfusion, which was fatal in some cases. MMP-2 deficiency resulted in upregulation of MMP-9 activity, spontaneous leukocyte infiltration in naïve livers, and amplified MMP-9-dependent transmigration of leukocytes in vitro and after hepatic IRI. Moreover, complete loss of MMP-2 activity impaired the degradation of poly (ADP-ribose) polymerase (PARP-1) in extensively damaged livers post-reperfusion. However, the administration of a PARP-1 inhibitor to MMP-2 null mice restored liver preservation to almost comparable levels of MMP-2+/+ mice post-IRI. Deficient PARP-1 degradation in MMP-2-null sinusoidal endothelial cells correlated with their increased cytotoxicity, evaluated by the measurement of LDH efflux in the medium. In conclusion, our results show for the first time that MMP-2 gene deletion exacerbates liver IRI. Moreover, they offer new insights into the MMP-2 modulation of inflammatory responses, which could be relevant for the design of new pharmacological MMP-targeted agents to treat hepatic IRI.

Tissue inhibitor of metalloproteinase (TIMP) 3 is a naturally occurring inhibitor of a broad range of proteases, with key roles in extracellular matrix turnover and in the pathogenesis of various diseases. In this study, we investigated the response of mice lacking TIMP3 (TIMP3-/-) to hepatic ischemia/reperfusion injury (IRI). We report here that TIMP3-/- mice showed an enhanced inflammatory response, exacerbated organ damage, and further impaired liver function after IRI when compared with their wild-type littermates. Loss of TIMP3 led to the cleavage and shedding of E-cadherin during hepatic IRI; the full-length 120-kDa E-cadherin and the ratio of 38-kDa C-terminal fragment/120-kDa E-cadherin were decreased and increased, respectively, in TIMP3-/- livers after IRI. Moreover, GI254023X, a potent inhibitor of a disintegrin and metalloprotease (ADAM) 10, was capable of partially rescuing the expression of E-cadherin in the TIMP3-null hepatocytes. The proteolysis of E-cadherin in the TIMP3-/- livers was also linked to the loss of β-catenin from the hepatocyte membranes and to an increased susceptibility to apoptosis after liver IRI. In a similar fashion, depression of the E-cadherin/β-catenin complex mediated by TIMP3 deletion and knockdown of β-catenin by small interfering RNA were both capable of inducing caspase activation in isolated hepatocytes subjected to H₂ O₂ oxidative stress. Hence, these results support a protective role for TIMP3 expression in sheltering the hepatocyte E-cadherin/β-catenin complex from proteolytic processing and inhibiting apoptosis after hepatic IRI.

Background & aims

Organ shortage has led to the use of steatotic livers in transplantation, despite their elevated susceptibility to ischemia/reperfusion injury (IRI). Matrix metalloproteinase-9 (MMP-9), an inducible gelatinase, is emerging as a central mediator of leukocyte traffic into inflamed tissues. However, its role in steatotic hepatic IRI has yet to be demonstrated.

Methods

We examined the function of MMP-9 in mice fed with a high-fat diet (HFD), which developed approximately 50% hepatic steatosis, predominantly macrovesicular, prior to partial hepatic IRI.

Results

The inability of MMP-9(-/-) deficient steatotic mice to express MMP-9 significantly protected these mice from liver IRI. Compared to fatty controls, MMP-9(-/-) steatotic livers showed significantly reduced leukocyte infiltration, proinflammatory cytokine expression, and liver necrosis. Loss of MMP-9 activity preserved platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, a modulator of vascular integrity at the endothelial cell-cell junctions in steatotic livers after IRI. Using in vitro approaches, we show that targeted inhibition of MMP-9 sheltered the extracellular portion of PECAM-1 from proteolytic processing, and disrupted leukocyte migration across this junctional molecule. Moreover, the evaluation of distinct parameters of regeneration, proliferating cell nuclear antigen (PCNA) and histone H3 phosphorylation (pH3), provided evidence that hepatocyte progression into S phase and mitosis was notably enhanced in MMP-9(-/-) steatotic livers after IRI.

Conclusions

MMP-9 activity disrupts vascular integrity at least partially through a PECAM-1 dependent mechanism and interferes with regeneration of steatotic livers after IRI. Our novel findings establish MMP-9 as an important mediator of steatotic liver IRI.

The purpose of this study was to assess the significance of tenascin-C (Tnc) expression in steatotic liver ischemia/reperfusion injury (IRI). The critical shortage in donor organs has led to the use of steatotic livers in transplantation regardless of their elevated susceptibility to hepatic IRI. Tnc is an endogenous danger signal extracellular matrix molecule involved in various aspects of immunity and tissue injury. In the current study, mice were fed with a steatosis-inducing diet and developed approximately 50% hepatic steatosis, predominantly macrovesicular, before being subjected to hepatic IRI. We report here that lipid accumulation in hepatocytes inflated the production of Tnc in steatotic livers and in isolated hepatic stellate cells. Moreover, we show that the inability of Tnc-/- deficient steatotic mice to express Tnc significantly protected these mice from liver IRI. Compared with fatty controls, Tnc-/- steatotic mice showed significantly reduced serum transaminase levels and enhanced liver histological preservation at both 6 and 24 hours after hepatic IRI. The lack of Tnc expression resulted in impaired lymphocyte antigen 6 complex, locus (Ly6G) neutrophil and macrophage antigen-1 (Mac-1) leukocyte recruitment as well as in decreased expression of proinflammatory mediators (interleukin 1β, tumor necrosis factor α, and chemokine [C-X-C motif] ligand 2) after liver reperfusion. Myeloperoxidase (MPO) is the most abundant cytotoxic enzyme secreted by neutrophils and a key mediator of neutrophil-induced oxidative tissue injuries. Using an in vitro model of steatosis, we also show that Tnc markedly potentiated the effect of steatotic hepatocytes on neutrophil-derived MPO activity. In conclusion, our data support the view that inhibition of Tnc is a promising therapeutic approach to lessen inflammation in steatotic livers and to maximize their successful use in organ transplantation.

BACKGROUND & AIMS: Organ shortage has led to the use of steatotic livers in transplantation, despite their elevated susceptibility to ischemia/reperfusion injury (IRI). Matrix metalloproteinase-9 (MMP-9), an inducible gelatinase, is emerging as a central mediator of leukocyte traffic into inflamed tissues. However, its role in steatotic hepatic IRI has yet to be demonstrated. METHODS: We examined the function of MMP-9 in mice fed with a high-fat diet (HFD), which developed approximately 50% hepatic steatosis, predominantly macrovesicular, prior to partial hepatic IRI. RESULTS: The inability of MMP-9(-/-) deficient steatotic mice to express MMP-9 significantly protected these mice from liver IRI. Compared to fatty controls, MMP-9(-/-) steatotic livers showed significantly reduced leukocyte infiltration, proinflammatory cytokine expression, and liver necrosis. Loss of MMP-9 activity preserved platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, a modulator of vascular integrity at the endothelial cell-cell junctions in steatotic livers after IRI. Using in vitro approaches, we show that targeted inhibition of MMP-9 sheltered the extracellular portion of PECAM-1 from proteolytic processing, and disrupted leukocyte migration across this junctional molecule. Moreover, the evaluation of distinct parameters of regeneration, proliferating cell nuclear antigen (PCNA) and histone H3 phosphorylation (pH3), provided evidence that hepatocyte progression into S phase and mitosis was notably enhanced in MMP-9(-/-) steatotic livers after IRI. CONCLUSIONS: MMP-9 activity disrupts vascular integrity at least partially through a PECAM-1 dependent mechanism and interferes with regeneration of steatotic livers after IRI. Our novel findings establish MMP-9 as an important mediator of steatotic liver IRI.

Cyclooxygenase-2 (COX-2) is a mediator of hepatic ischemia and reperfusion injury (IRI). While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M) to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9) expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s) other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

Cyclooxygenase-2 (COX-2) is a mediator of hepatic ischemia and reperfusion injury (IRI). While both global COX-2 deletion and pharmacologic COX-2 inhibition ameliorate liver IRI, the clinical use of COX-2 inhibitors has been linked to increased risks of heart attack and stroke. Therefore, a better understanding of the role of COX-2 in different cell types may lead to improved therapeutic strategies for hepatic IRI. Macrophages of myeloid origin are currently considered to be important sources of the COX-2 in damaged livers. Here, we used a Cox-2flox conditional knockout mouse (COX-2-M/-M) to examine the function of COX-2 expression in myeloid cells during liver IRI. COX-2-M/-M mice and their WT control littermates were subjected to partial liver ischemia followed by reperfusion. COX-2-M/-M macrophages did not express COX-2 upon lipopolysaccharide stimulation and COX-2-M/-M livers showed reduced levels of COX-2 protein post-IRI. Nevertheless, selective deletion of myeloid cell-derived COX-2 failed to ameliorate liver IRI; serum transaminases and histology were comparable in both COX-2-M/-M and WT mice. COX-2-M/-M livers, like WT livers, developed extensive necrosis, vascular congestion, leukocyte infiltration and matrix metalloproteinase-9 (MMP-9) expression post-reperfusion. In addition, myeloid COX-2 deletion led to a transient increase in IL-6 levels after hepatic reperfusion, when compared to controls. Administration of celecoxib, a selective COX-2 inhibitor, resulted in significantly improved liver function and histology in both COX-2-M/-M and WT mice post-reperfusion, providing evidence that COX-2-mediated liver IRI is caused by COX-2 derived from a source(s) other than myeloid cells. In conclusion, these results support the view that myeloid COX-2, including myeloid-macrophage COX-2, is not responsible for the hepatic IRI phenotype.

Foxo1 transcription factor is an evolutionarily conserved regulator of cell metabolism, oxidative stress, inflammation, and apoptosis. Activation of Hedgehog/Gli signaling is known to regulate cell growth, differentiation, and immune function. However, the molecular mechanisms by which interactive cell signaling networks restrain oxidative stress response and necroptosis are still poorly understood. Here, we report that myeloid-specific Foxo1 knockout (Foxo1^M-KO) mice were resistant to oxidative stress-induced hepatocellular damage with reduced macrophage/neutrophil infiltration, and proinflammatory mediators in liver ischemia/reperfusion injury (IRI). Foxo1^M-KO enhanced β-catenin-mediated Gli1/Snail activity, and reduced receptor-interacting protein kinase 3 (RIPK3) and NIMA-related kinase 7 (NEK7)/NLRP3 expression in IR-stressed livers. Disruption of Gli1 in Foxo1^M-KO livers deteriorated liver function, diminished Snail, and augmented RIPK3 and NEK7/NLRP3. Mechanistically, macrophage Foxo1 and β-catenin colocalized in the nucleus, whereby the Foxo1 competed with T-cell factor (TCF) for interaction with β-catenin under inflammatory conditions. Disruption of the Foxo1-β-catenin axis by Foxo1 deletion enhanced β-catenin/TCF binding, activated Gli1/Snail signaling, leading to inhibited RIPK3 and NEK7/NLRP3. Furthermore, macrophage Gli1 or Snail knockout activated RIPK3 and increased hepatocyte necroptosis, while macrophage RIPK3 ablation diminished NEK7/NLRP3-driven inflammatory response. Our findings underscore a novel molecular mechanism of the myeloid Foxo1-β-catenin axis in regulating Hedgehog/Gli1 function that is key in oxidative stress-induced liver inflammation and necroptosis.