Constantino Fondevila

Biliverdin therapy protects rat livers from ischemia and reperfusion injury

Heme oxygenase (HO‐1) provides a cellular defense mechanism during oxidative stress and catalyzes the rate‐limiting step in heme metabolism that produces biliverdin (BV). The role of BV and its potential use in preventing ischemia/reperfusion injury (IRI) had never been studied. This study was designed to explore putative cytoprotective functions of BV during hepatic IRI in rat liver models of ex vivo perfusion and orthotopic liver transplantation (OLT) after prolonged periods of cold ischemia. In an ex vivo hepatic IRI model, adjunctive BV improved portal venous blood flow, increased bile production, and decreased hepatocellular damage. These findings were correlated with amelioration of histological features of IRI, as assessed by Suzukis criteria. Following cold ischemia and syngeneic OLT, BV therapy extended animal survival from 50% in untreated controls to 90% to 100%. This effect correlated with improved liver function and preserved hepatic architecture. Additionally, BV adjuvant after OLT decreased endothelial expression of cellular adhesion molecules (P‐selectin and intracellular adhesion molecule 1), and decreased the extent of infiltration by neutrophils and inflammatory macrophages. BV also inhibited expression of inducible nitric oxide synthase and proinflammatory cytokines (interleukin 1β, tumor necrosis factor α, and interleukin 6) in OLTs. Finally, BV therapy promoted an increased expression of antiapoptotic molecules independently of HO‐1 expression, consistent with BV being an important mediator through which HO‐1 prevents cell death. In conclusion, this study documents and dissects potent cytoprotective effects of BV in well‐established rat models of hepatic IRI. Our results provide the rationale for a novel therapeutic approach using BV to maximize the function and thus the availability of donor organs. (HEPATOLOGY 2004;40:1333–1341.)

Metalloproteinase‐9 deficiency protects against hepatic ischemia/reperfusion injury

Leukocyte transmigration across endothelial and extracellular matrix protein barriers is dependent on adhesion and focal matrix degradation events. In the present study we investigated the role of metalloproteinase‐9 (MMP‐9/gelatinase B) in liver ischemia/reperfusion (I/R) injury using MMP‐9‐deficient (MMP‐9−/−) animals and mice treated with a specific anti‐MMP‐9 neutralizing antibody or with a broad gelatinase inhibitor for both MMP‐9 and metalloproteinase‐2 (MMP‐2/gelatinase A). Compared to wild‐type mice, MMP‐9−/− mice and mice treated with an anti‐MMP‐9 antibody showed significantly reduced liver damage. In contrast, mice treated with a broad gelatinase inhibitor showed rather inferior protection against I/R injury and were characterized by persistent ongoing liver inflammation, suggesting that MMP‐2 and MMP‐9 may have distinct roles in this type of injury. MMP‐9 was mostly detected in Ly‐6G and macrophage antigen–1 leukocytes adherent to the vessel walls and infiltrating the damaged livers of wild‐type mice after liver I/R injury. Leukocyte traffic and cytokine expression were markedly impaired in livers of MMP‐9−/− animals and in livers of mice treated with anti‐MMP‐9 antibody after I/R injury; however, initiation of the endothelial adhesion cascades was similar in both MMP‐9−/− and control livers. We also showed that MMP‐9‐specific inhibition disrupted neutrophil migration across fibronectin in transwell filters and depressed myeloperoxidase (MPO) activation in vitro. Conclusion: These results support critical functions for MMP‐9 in leukocyte recruitment and activation leading to liver damage. Moreover, they provide the rationale for identifying inhibitors to specifically target MMP‐9 in vivo as a potential therapeutic approach in liver I/R injury. (HEPATOLOGY 2007.)

Molecular characterization of rat leukocyte P-selectin glycoprotein ligand-1 and effect of its blockade: protection from ischemia-reperfusion injury in liver transplantation.

P-selectin glycoprotein ligand-1 (PSGL-1) mediates the initial tethering of leukocytes to activated platelets and endothelium. We report molecular cloning and characterization of the rat PSGL-1 gene. A neutralizing Ab was generated, and its binding epitope was mapped to the N-terminal binding region of rat PSGL-1. We examined the effects of early PSGL-1 blockade in rat liver models of cold ischemia, followed by ex vivo reperfusion or transplantation (orthotopic liver transplantation (OLT)) using an anti-PSGL-1 Ab with diminished Fc-mediated effector function. In the ex vivo hepatic cold ischemia and reperfusion model, pretreatment with anti-PSGL-1 Ab improved portal venous flow, increased bile production, and decreased hepatocellular damage. Rat pretreatment with anti-PSGL-1 Ab prevented hepatic insult in a model of cold ischemia, followed by OLT, as assessed by 1) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic-pyruvic transaminase levels), and ameliorated histological features of ischemia/reperfusion injury, consistent with extended OLT survival; 2) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin, ED-1, CD3, and OX-62 cells; 3) inhibited expression of proinflammatory cytokine genes (TNF-α, IL-1β, IL-6, IFN-γ, and IL-2); and 4) prevented hepatic apoptosis accompanied by up-regulation of antiapoptotic Bcl-2/Bcl-xL protective genes. Thus, targeting PSGL-1 with a blocking Ab that has diminished Fc-mediated effector function is a simple and effective strategy that provides the rationale for novel therapeutic approaches to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.

Decompression of the portal bed and twice-baseline portal inflow are necessary for the functional recovery of a "small-for-size" graft.

Background.In partial liver transplant, a reduction in the intrahepatic vascular bed produces a rise in the portal vein flow and the portal venous pressure gradient, leading to endothelial and, thereby, hepatocellular injury and death in a process known as “small-for-size” (SFS) syndrome. Objective.To demonstrate that a calibrated portocaval shunt prevents superfluous inflow in a porcine model of SFS transplant. Methods.Donor pigs (15–20 kg) underwent 70% hepatectomy. In 2 groups, a 6 mm (S6) (n = 6) or 12 mm (S12) (n = 6) Gore-Tex shunt was placed between the portal vein and infrahepatic inferior vena cava. In a third group, no portocaval shunt was placed (SFS) (n = 17). Grafts were stored for 5 hours at 4°C and then transplanted into recipients (30–35 kg). Results.Five-day survival was 29% in SFS, 100% in S6, and 0 in S12. Postreperfusion portal vein flow was 4-, 2-, and 1-times flow at baseline in SFS, S6, and S12, respectively. With respect to portal venous pressure gradient, both the 6- and 12-mm shunts effectively decompressed the portal bed. Aspartate aminotransferase and bilirubin rose and the Quick prothrombin time fell in all animals after reperfusion but improved significantly by day 5 in S6. Serum levels of endothelin-1 remained elevated in SFS and S12 but returned to baseline by 12 hours in S6: 2.76 (2.05–4.08) and 2.04 (1.97–2.12) versus 0.43 (0.26–0.50) pg/mL, respectively (P < 0.05 for both comparisons). Conclusions.A calibrated portocaval shunt that maintains portal vein flow about twice its baseline value produces a favorable outcome after SFS liver transplantation, avoiding endothelial injury due to portal hyperperfusion or to hypoperfusion because of excess shunting.

In-vivo normothermic recirculation: an update.

Purpose of reviewThe use of nonheart-beating donors (NHBD), as a source for liver grafts, is the only way to objectively increase the activity in liver transplantation. Through the use of more strict criteria, 1-year graft survival has increased from 50% in initial series to 84–100%. With respect to type II NHBD (failure of cardiopulmonary resuscitation) we believe that the use of normothermic recirculation provides the possibility to obtain good quality grafts for transplantation. Our recent experience shows a 66% and 73% graft and patient survival respectively. Recent findingsHowever, the incidence of biliary tract complications (ischemic type), as well as the relative low number of grafts procured that are finally transplanted, makes this methodology difficult to develop. The use of a ‘normothermic machine perfusion’ (once the liver is procured), added to the already described method, may actually increase the efficacy and safety of the whole procedure. Recent experience by our group as well as others have shown that the liver so perfused is able to recover from the warm ischemic lesion and at the same time the procedure allows the possibility to evaluate the quality of the potential graft. SummaryNHBD are the only source that may objectively increased the number of liver transplant. The use of normothermic recirculation has proved to be effective and well tolerated. The addition of the ‘normothermic machine perfusion’ to the whole procedure may significantly increase the number of transplants.

The membrane attack complex (C5b‐9) in liver cold ischemia and reperfusion injury

Activation of the complement cascade represents an important event during ischemia/reperfusion injury (IRI). This work was designed to investigate the role of the membrane attack complex (MAC; C5b‐9) in the pathogenesis of hepatic IRI. Livers from B&W/Stahl/rC6(+) and C6(−) rats were harvested, stored for 24 hours at 4°C, and then transplanted [orthotopic liver transplantation (OLT)] to syngeneic recipients. There were 4 experimental groups: (1) C6(+)→C6(+), (2) C6(+)→C6(−), (3) C6(−)→C6(+), and (4) C6(−)→C6(−). At day +1, C6(−) OLTs showed decreased vascular congestion/necrosis, contrasting with extensive necrosis in C6(+) livers, that was independent of the recipient C6 status (Suzuki score: 7.2 ± 0.9, 7.3 ± 1.3, 4.5 ± 0.6, and 4.8 ± 0.4 for groups 1‐4, respectively, P < 0.05). The liver function improved in recipients of C6(−) grafts (serum glutamic oxaloacetic transaminase: 2573 ± 488, 1808 ± 302, 1170 ± 111, and 1188 ± 184 in groups 1‐4, respectively, P < 0.05). Intragraft macrophage infiltration (ED‐1 immunostaining) and neutrophil infiltration (myeloperoxidase activity) were reduced in C6(−) grafts versus C6(+) grafts (P = 0.001); these data were confirmed by esterase staining (naphthol). The expression of proinflammatory interferon‐γ, interleukin‐1β, and tumor necrosis factor messenger RNA/protein was also reduced in C6(−) OLTs in comparison with C6(+) OLTs. Western blot–assisted expression of proapoptotic caspase‐3 was decreased in C6(−) OLTs versus C6(+) OLTs (P = 0.006), whereas antiapoptotic Bcl‐2/Bag‐1 was enhanced in C6(−) OLTs compared with C6(+) OLTs (P = 0.001). Terminal deoxynucleotidyl transferase–mediated dUTP nick end‐labeling staining of apoptotic cells was enhanced (P < 0.05) in C6(+) OLTs compared with C6(−) OLTs. Thus, the terminal products of the complement system are essential in the mechanism of hepatic IRI. This is the first report using a clinically relevant liver cold ischemia model to show that local MAC inhibition attenuates IRI cascade in OLT recipients. Liver Transpl 14:1133–1141, 2008.

Extracorporeal machine liver perfusion: are we warming up?

Purpose of reviewRecently, considerable focus has been placed on the use of hypothermic perfusion ex vivo in abdominal organ transplant. Herein, we discuss the appropriateness of using this modality to preserve livers, in particular those of suboptimal quality, and whether perfusing at warmer temperatures in this context may, in fact, be better. Recent findingsHypothermic perfusion (0–4°C) appears to improve the hepatocellular energy charge and achieve adequate results in normal livers. However, its use for the preservation of suboptimal grafts may lead to significant endothelial and Kupffer cell injury that is incompatible with survival. Studies on the perfusion of suboptimal livers at higher temperatures, on the contrary, indicate that results improve as temperatures approach 37°C, provided that the oxygen supply during perfusion is adequate. SummaryNormothermic perfusion provides oxygen and other metabolic substrates under physiological conditions; in liver transplant, it appears to be the best option to improve the viability of suboptimal organs.

Biliary complications after adult living donor liver transplantation

We evaluated the causes and outcomes of biliary complications occurring after adult living donor liver transplantation (ALDLT) in a large patient cohort. Among 46 patients who underwent ALDLT at two different centers early bile duct complications occurred in 11 recipients (23.9%), consisting of leakage from the anastomotic site or from the cut surface of the liver. T-tube-associated biliary complications occurred in four patients. Late complications, primarily anastomotic strictures, occurred in 15 patients (32.6%) at 6.7+/-3.5 months after transplantation. Surgical intervention was generally required for early biliary complications but rarely necessary for late complications. No graft loss was caused by biliary complications. Thus, ALDLT is accompanied by a high rate of biliary complications, which in our series have been of low severity. However, long-term effects on graft function are not yet known.

Biliverdin protects rat livers from ischemia/reperfusion injury.

OBJECTIVE To explore putative cytoprotective functions of biliverdin during hepatic ischemia/reperfusion (I/R) injury in rat models. MATERIAL AND METHODS Male Sprague Dawley (SD) rat livers were harvested and stored for 24 hours at 4 degrees C in University of Wisconsin (UW) solution (n=18), and then perfused with blood for 2 hours on an isolated rat liver perfusion apparatus equipped for temperature (37 degrees C), pressure (13 cm H2O), and pH (7.3) maintenance. Biliverdin was added to the blood at concentrations of 10 and 50 micromol in two groups of six animals. Portal vein blood flow, bile production, and GOT/GPT levels were assessed serially. At the conclusion of the experiment, liver samples were collected for histologic evaluation using Suzuki criteria. RESULTS BV exerted protective effects against liver I/R injury. Adjunctive biliverdin improved portal venous blood flow (mL/min/g) from the beginning of reperfusion (1.33+/-0.17 versus 0.98+/-0.15; P<.001) and increased bile production (mL/g) as compared with the control group (3.40 versus 1.88; P<.003). I/R-induced hepatocellular damage as measured by GOT/GPT release (IU/L) was diminished in the biliverdin group (91 versus 171 and 46 versus 144, respectively; P<.0001). Improved liver function by biliverdin was accompanied by preservation of the histologic structure as assessed by Suzuki criteria (3.7+/-1.4 versus 6.8+/-0.8 in untreated controls; P<.005). CONCLUSIONS Biliverdin attenuates the ischemia/early reperfusion injury of rat liver grafts as assessed by hemodynamics, function, enzyme analysis, and histology. This study provides the rationale for novel therapeutic approaches using biliverdin to maximize the organ donor pool through the safer use of liver transplants despite prolonged periods of cold ischemia.

Outcome of patients following hepatic resection for metastatic cutaneous and ocular melanoma

Background/purposeThe aim of this study was to analyze the outcome of patients undergoing hepatic resection for melanoma liver metastases.MethodsPatients undergoing liver resection for melanoma metastases at the Hospital Vall d’Hebron and Hospital Clinic, Barcelona, were reviewed. Selection criteria were: good performance status, feasibly complete and safe resection, and absence of visceral extrahepatic metastases.ResultsBetween 1994 and 2007, 14 liver resections were performed for melanoma liver metastases. The primary tumor was cutaneous in 8 patients and ocular in 6. Two patients underwent urgent liver surgery due to tumor bleeding. In these patients, complete melanoma staging was not performed and extrahepatic metastases were found during surgery or during the postoperative course. Six of 13 patients (46.2%) developed liver recurrence during follow-up. One- and 3-year actuarial patient survivals were 77 and 49%, respectively. Excluding the patients who underwent urgent liver surgery, the 1- and 3-year actuarial patient survivals in those with primary ocular and cutaneous melanoma were 83 and 56% and 80 and 60%, respectively.ConclusionsLiver resection may be considered as part of oncosurgical treatment in patients with melanoma liver metastases, since prolonged survival was observed, albeit with a high recurrence rate. Nevertheless, it should be taken into account that our study included only a small number of patients.