Sergio Duarte

Matrix metalloproteinases in liver injury, repair and fibrosis

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.

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

Cyclooxygenase-2 (COX-2) is a prostanoid-synthesizing enzyme that is critically implicated in a variety of pathophysiological processes. Using a COX-2-deficient mouse model, we present data that suggest that COX-2 has an active role in liver ischemia/reperfusion (I/R) injury. We demonstrate that COX-2-deficient mice had a significant reduction in liver damage after I/R insult. The inability of COX-2−/− to elaborate COX-2 products favored a Th2-type response in these mice. COX-2−/− livers after I/R injury showed significantly decreased levels of IL-2, as well as IL-12, a cytokine known to have a central role in Th1 effector cell differentiation. Moreover, such livers expressed enhanced levels of the anti-inflammatory cytokine IL-10, shifting the balance in favor of a Th2 response in COX-2-deficient mice. The lack of COX-2 expression resulted in decreased levels of CXCL2, a neutrophil-activating chemokine, reduced infiltration of MMP-9-positive neutrophils, and impaired late macrophage activation in livers after I/R injury. Additionally, Bcl-2 and Bcl-xL were normally expressed in COX-2−/− livers after injury, whereas respective wild-type controls were almost depleted of these two inhibitors of cell death. In contrast, caspase-3 activation and TUNEL-positive cells were depressed in COX-2−/− livers. Therefore, our data support the concept that COX-2 is involved in the pathogenic events occurring in liver I/R injury. The data also suggest that potential valuable therapeutic approaches in liver I/R injury may result from further studies aimed at identifying specific COX-2-derived prostanoid pathways.

Inducible nitric oxide synthase deficiency impairs matrix metalloproteinase-9 activity and disrupts leukocyte migration in hepatic ischemia/reperfusion injury.

Matrix metalloproteinase 9 (MMP-9) is a critical mediator of leukocyte migration in hepatic ischemia/reperfusion (I/R) injury. To test the relevance of inducible nitric oxide synthase (iNOS) expression on the regulation of MMP-9 activity in liver I/R injury, our experiments included both iNOS-deficient mice and mice treated with ONO-1714, a specific iNOS inhibitor. The inability of iNOS-deficient mice to generate iNOS-derived nitric oxide (NO) profoundly inhibited MMP-9 activity and depressed leukocyte migration in livers after I/R injury. While macrophages expressed both iNOS and MMP-9 in damaged wild-type livers, neutrophils expressed MMP-9 and were virtually negative for iNOS; however, exposure of isolated murine neutrophils and macrophages to exogenous NO increased MMP-9 activity in both cell types, suggesting that NO may activate MMP-9 in leukocytes by either autocrine or paracrine mechanisms. Furthermore, macrophage NO production through the induction of iNOS was capable of promoting neutrophil transmigration across fibronectin in a MMP-9-dependent manner. iNOS expression in liver I/R injury was also linked to liver apoptosis, which was reduced in the absence of MMP-9. These results suggest that MMP-9 activity induced by iNOS-derived NO may also lead to detachment of hepatocytes from the extracellular matrix and cell death, in addition to regulating leukocyte migration across extracellular matrix barriers. These data provide evidence for a novel mechanism by which MMP-9 can mediate iNOS-induced liver I/R injury.

Tenascin‐C: A novel mediator of hepatic ischemia and reperfusion injury

Hepatic ischemia/reperfusion (IRI) injury remains a major challenge in clinical orthotopic liver transplantation (OLT). Tenascin‐C (Tnc) is an extracellular matrix protein (ECM) involved in various aspects of immunity and tissue injury. Using a Tnc‐deficient mouse model, we present data that suggest an active role for Tnc in liver IRI. We show that Tnc‐deficient mice have a reduction in liver damage and a significant improvement in liver regeneration after IRI. The inability of Tnc−/− mice to express Tnc significantly reduced the levels of active caspase‐3/transferase‐mediated dUTP nick end‐labeling (TUNEL) apoptotic markers and enhanced the expression of the proliferation cell nuclear antigen (PCNA) after liver IRI. The lack of Tnc expression resulted in impaired leukocyte recruitment and decreased expressions of interleukin (IL)‐1β, IL‐6, and CXCL2 after liver reperfusion. Tnc‐deficient livers were characterized by altered expression patterns of vascular adhesion molecules, such as vascular cell adhesion molecule‐1 and platelet endothelial cell adhesion molecule‐1 post‐IRI. Moreover, matrix metalloproteinase‐9 (MMP‐9) synthesis, which facilitates leukocyte transmigration across vascular barriers in liver IRI, was markedly down‐regulated in the absence of Tnc. We also show that Tnc is capable of inducing MMP‐9 expression in isolated neutrophils through Toll‐like receptor 4. Therefore, our data suggest that Tnc is a relevant mediator of the pathogenic events underlying liver IRI. The data also support the view that studies aimed at further understanding how newly synthesized ECM molecules, such as Tnc, participate in inflammatory responses are needed to improve therapeutic approaches in liver IRI. (HEPATOLOGY 2011)

TIMP-1 deficiency leads to lethal partial hepatic ischemia and reperfusion injury†‡

Hepatic ischemia and reperfusion injury (IRI) remains an important challenge in clinical orthotopic liver transplantation (OLT). Tissue inhibitor of metalloproteinase‐1 (TIMP‐1) is the major endogenous regulator of matrix metalloproteinase‐9 (MMP‐9). In this study we investigated the functional significance of TIMP‐1 expression in a well‐established mouse model of partial liver IRI. Compared to wildtype mice, TIMP‐1−/− mice showed further impaired liver function and histological preservation after IRI. Notably, TIMP‐1 deficiency led to lethal liver IRI, as over 60% of the TIMP‐1−/− mice died postreperfusion, whereas all TIMP‐1+/+ mice recovered and survived surgery. Lack of TIMP‐1 expression was accompanied by markedly high levels of MMP‐9 activity, which facilitates leukocyte transmigration across vascular barriers in hepatic IRI. Indeed, TIMP‐1−/− livers were characterized by massive leukocyte infiltration and by up‐regulation of proinflammatory mediators, including tumor necrosis factor alpha, interferon‐gamma, and inducible nitric oxide synthase post‐IRI. The inability of TIMP‐1−/− mice to express TIMP‐1 increased the levels of active caspase‐3 and depressed the expression of Bcl‐2 and the phosphorylation of Akt, emphasizing an important role for TIMP‐1 expression on hepatocyte survival. Using independent parameters of regeneration, 5‐bromodeoxyuridine incorporation, proliferating cell nuclear antigen expression, and histone H3 phosphorylation, we provide evidence that hepatocyte progression into S phase and mitosis was impaired in TIMP‐1‐deficient livers after IRI. Inhibition of the cell cycle progression by TIMP‐1 deficiency was linked to depressed levels of cyclins‐D1 and ‐E and to a disrupted c‐Met signaling pathway, as evidenced by reduced phosphorylated c‐Met expression and elevated c‐Met ectodomain shedding postliver IRI. Conclusion: These results support a critical protective function for TIMP‐1 expression on promoting survival and proliferation of liver cells and on regulating leukocyte recruitment and activation in liver IRI. (HEPATOLOGY 2012;56:1074–1085)

MMP-9 deficiency shelters endothelial PECAM-1 expression and enhances regeneration of steatotic livers after ischemia and reperfusion injury.

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.

Cytoprotective Effects of a Cyclic RGD Peptide in Steatotic Liver Cold Ischemia and Reperfusion Injury

The serious need for expanding the donor population has attracted attention to the use of steatotic donor livers in orthotopic liver transplantation (OLT). However, steatotic livers are highly susceptible to hepatic ischemia–reperfusion injury (IRI). Expression of fibronectin (FN) by endothelial cells is an important feature of hepatic response to injury. We report the effect of a cyclic RGD peptide with high affinity for the α5β1, the FN integrin receptor, in a rat model of steatotic liver cold ischemia, followed by transplantation. RGD peptide therapy ameliorated steatotic IRI and improved the recipient survival rate. It significantly inhibited the recruitment of monocyte/macrophages and neutrophils, and depressed the expression of pro‐inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and interferon (IFN)‐γ. Moreover, it resulted in profound inhibition of metalloproteinase‐9 (MMP‐9) expression, a gelatinase implied in leukocyte migration in damaged livers. Finally, we show that RGD peptide therapy reduced the expression of the 17‐kDa active caspase‐3 and the number of apoptotic cells in steatotic OLTs. The observed protection against steatotic liver IRI by the cyclic RGD peptides with high affinity for the α5β1 integrin suggests that this integrin is a potential therapeutic target to allow the successful utilization of marginal steatotic livers in transplantation.

Fibronectin-α4β1 Interactions in Hepatic Cold Ischemia and Reperfusion Injury: Regulation of MMP-9 and MT1-MMP via the p38 MAPK Pathway

Liver ischemia‐reperfusion injury (IRI) remains a challenging problem in clinical settings. The expression of fibronectin (FN) by endothelial cells is a prominent feature of the hepatic response to injury. Here we investigate the effects of the connecting segment‐1 (CS‐1) peptide therapy, which blocks FN‐α4β1 integrin leukocyte interactions, in a well‐established model of 24‐h cold liver IRI. CS‐1 peptides significantly inhibited leukocyte recruitment and local release of proinflammatory mediators (COX‐2, iNOS and TNF‐α), ameliorating liver IRI and improving recipient survival rate. CS1 therapy inhibited the phosphorylation of p38 MAPK, a kinase linked to inflammatory processes. Moreover, in addition to downregulating the expression of matrix metalloproteinase‐9 (MMP‐9) in hepatic IRI, CS‐1 peptide therapy depressed the expression of membrane type 1‐matrix metalloproteinase (MT1‐MMP/MMP‐14) by macrophages, a membrane‐tethered MMP important for focal matrix proteolysis. Inhibition of p38 MAPK activity, with its pharmacological antagonist SB203580, downregulated MMP‐9 and MT1‐MMP/MMP‐14 expressions by FN‐stimulated macrophages, suggesting that p38 MAPK kinase pathway controls FN‐mediated inductions of MMP‐9 and MT1‐MMP/MMP‐14. Hence, this study provides new insights on the role of FN in liver injury, which can potentially be applied to the development of new pharmacological strategies for the successful protection against hepatic IRI.

Matrix Metalloproteinase-2 (MMP-2) Gene Deletion Enhances MMP-9 Activity, Impairs PARP-1 Degradation, and Exacerbates Hepatic Ischemia and Reperfusion Injury in Mice

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.

Aminoacylase 3 Is a New Potential Marker and Therapeutic Target in Hepatocellular Carcinoma

Ras proteins (HRas, KRas and NRas) are common oncogenes that require membrane association for activation. Previous approaches to block/inhibit Ras membrane association were unsuccessful for cancer treatment in human clinical studies. In the present study we utilized a new approach to decrease Ras membrane association in hepatocellular carcinoma (HCC) cell lines via inhibition of an enzyme aminoacylase 3 (AA3; EC 3.5.1.114). AA3 expression was significantly elevated in the livers of HCC patients and HCC cell lines. Treatment of HepG2 cells with AA3 inhibitors, and HepG2 and HuH7 with AA3 siRNA significantly decreased Ras membrane association and was toxic to these HCC cell lines. AA3 inhibitors also increased the levels of N-acetylfarnesylcysteine (NAFC) and N-acetylgeranylgeranylcysteine (NAGGC) in HepG2 and Huh7 cell lines. We hypothesized that AA3 deacetylates NAFC and NAGGC, and generated farnesylcysteine (FC) and geranylgeranylcysteine (GGC) that are used in HCC cells for the regeneration of farnesylpyrophosphate and geranylgeranylpyrophosphate providing the prenyl (farnesyl or geranylgeranyl) group for Ras prenylation required for Ras membrane association. This was confirmed experimentally where purified human AA3 was capable of efficiently deacetylating NAFC and NAGGC. Our findings suggest that AA3 inhibition may be an effective approach in the therapy of HCC and that elevated AA3 expression in HCC is potentially an important diagnostic marker.