Ian B. Nicoud

Warm hepatic ischemia-reperfusion promotes growth of colorectal carcinoma micrometastases in mouse liver via matrix metalloproteinase-9 induction

Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromal-derived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery.

Silencing of TLR4 Decreases Liver Tumor Burden in a Murine Model of Colorectal Metastasis and Hepatic Steatosis

BackgroundThe relationship between obesity and cancer has become of particular interest due to the rapidly growing prevalence of overweight individuals. Obesity predisposes individuals to the development of hepatic steatosis and is an independent risk factor for several neoplasms. Toll-like receptor 4 (TLR4) is the innate receptor for endotoxin, and steatotic livers are known to be sensitive to endotoxin. TLR4 signaling has been shown to have proneoplastic effects in vitro due to its effect on immune surveillance. Thus far, studies have predominantly focused on the effect of tumor-cell-derived TLR4 without regard to host TLR4 signaling.ResultsIn the present study we show that steatotic livers have increased expression of TLR4. Obese animals developed higher metastatic tumor burden in the liver than lean controls regardless of the presence or absence of intact host TLR4. After silencing TLR4 expression using RNAi in the mouse colon cancer cell line MC38, there was a significant decrease in metastatic tumor burden within the liver of obese animals.ConclusionsThese findings demonstrate that steatotic livers have increased susceptibility to metastatic tumor growth and that silencing tumor cell TLR4 reduces metastatic tumor burden in steatotic liver.

Modulation of mitochondrial calcium management attenuates hepatic warm ischemia-reperfusion injury.

Hepatic warm ischemia and reperfusion (IR) injury occurs in many clinical situations and has an important link to subsequent hepatic failure. The pathogenesis of this injury involves numerous pathways, including mitochondrial‐associated apoptosis. We studied the effect of mitochondrial calcium uptake inhibition on hepatic IR injury using the specific mitochondrial calcium uptake inhibitor, ruthenium red (RR). Rats were subjected to 1 hour of 70% warm hepatic ischemia following RR pretreatment or vehicle injection. Sham‐operated animals served as controls. Analysis was performed at 15 minutes, 1 hour, 3 hours, or 6 hours after reperfusion. Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) concentrations were determined. Terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling (TUNEL) staining was performed to assess apoptosis, and hepatocellular necrosis was semiquantitated from hematoxylin and eosin–stained tissue sections. RR pretreatment significantly decreased both AST and ALT serum levels after 6 hours of reperfusion (AST: 1,556 ± 181 U/L vs. 597 ± 121 U/L, P = 0.005; ALT: 1,118 ± 187 U/L vs. 294 ± 39 U/L, P = 0.005). Apoptosis was observed within 15 minutes of reperfusion in vehicle‐pretreated animals and peaked after 3 hours of reperfusion (98 ± 21 cells/high‐power field [hpf]). Apoptosis was inhibited at all time points by RR pretreatment. Histologic evidence of necrosis was not observed prior to 3 hours of reperfusion (23% ± 4%), and maximal necrosis was observed after 6 hours of reperfusion (26% ± 1% percent area). RR pretreatment significantly decreased the necrotic percent area at both the 3‐hour and the 6‐hour time points (4.2% ± 2%; 3.7% ± 1%, respectively). Hepatic IR injury resulted in both apoptotic and necrotic cell death, which were attenuated by RR pretreatment. In conclusion, these observations implicate mitochondrial calcium uptake in the pathogenesis of hepatic IR injury. (Liver Transpl 2005;11:663–668.)

Mitochondrial Calcium Uptake Regulates Cold Preservation‐Induced Bax Translocation and Early Reperfusion Apoptosis

Mitochondrial calcium (mCa + 2) overload occurs during cold preservation and is an integral part of mitochondrial‐dependent apoptotic pathways. We investigated the role of mCa + 2 overload in cell death following hypothermic storage using HepG2 cells stored in normoxic‐hypothermic (4 °C) or hypoxic (< 0.1% O2)‐hypothermic Belzer storage solution. Cells were stored for 6 h, with or without 10 μM ruthenium red (mCa + 2 uniporter inhibitor) followed by rewarming in oxygenated media at 37 °C. Cytoplasmic cytochrome c levels were studied by Western analysis and by fluorescent microscopy after transfection of cytochrome c‐GFP expression plasmid. Immunofluorescence determined the intracellular, spatio‐temporal distribution of Bax, and TUNEL staining was used to evaluate cell death after 180 min of rewarming. Caspase activation was evaluated using Western analysis and a caspase 3 activity assay. Bax translocation, cytochrome c release, and early rewarming cell death occurred following hypothermic storage and were exacerbated by hypoxia. Caspase 3 activation did not occur following hypothermic storage. Blockade of mCa + 2 uptake prevented Bax translocation, cytochrome c release, and early rewarming cell death. These studies demonstrate that mCa + 2 uptake during hypothermic storage, both hypoxic and normoxic, contributes to early rewarming apoptosis by triggering Bax translocation to mitochondria and cytochrome c release.

Mitochondrial P2Y-Like receptors link cytosolic adenosine nucleotides to mitochondrial calcium uptake.

ATP is a known extracellular ligand for cell membrane purinergic receptors. Intracellular ATP can work also as a regulatory ligand via binding sites on functional proteins. We report herein the existence of P2Y1‐like and P2Y2‐like receptors in hepatocyte mitochondria (mP2Y1 and mP2Y2), which regulate mCa2+ uptake though the uniporter. Mitochondrial P2Y1 activation stimulates mCa2+ uptake; whereas, mP2Y2 activation inhibits mCa2+ uptake. ATP acts preferentially on mP2Y2 receptors, while ADP and AMP‐PNP stimulate both the mP2Y1 and mP2Y2. PPADS inhibits ADP stimulated mP2Y1‐mediated mCa2+ uptake. In addition, UTP, a selective P2Y2 agonist, strongly inhibits mCa2+ uptake. The newly discovered presence and function of these receptors is significant because it explains increased mCa2+ uptake in the setting of low cytosolic [ATP] and, therefore, establishes a mechanism for direct feedback in which cytosolic [ATP] governs mitochondrial ATP production through regulation of mCa2+ uptake.

Real-time spectroscopic assessment of thermal damage: implications for radiofrequency ablation

Radiofrequency ablation (RFA) is an evolving technology used to treat unresectable liver tumors. Currently, there is no accurate method to determine RFA margins in real-time during the procedure. We hypothesized that fiber-optic based spectroscopic monitoring system could detect thermal damage from RFA in real-time. Fluorescence (F) and diffuse reflectance (Rd) spectra were continuously acquired from within the expected ablation zone during canine hepatic RFA using a fiber-optic microinterrogation probe (MIP). The F and Rd spectral feedback were continuously monitored and ablations were stopped based on changes in spectra alone. After each ablation, the MIP tract was marked with India ink and the ablation zone was excised. The relationship of the MIP to the zone of ablation was examined grossly and microscopically. F and Rd spectral changes occurred in three characteristic phases as the ablation zone progresses past the MIP. Phase 1 indicates minimal deviation from normal lives. Phase 2 occurs as the MIP lies within the hemorrhagic zone of the ablated tissue. Phase 3 correlates with complete tissue coagulation. The absolute magnitude of spectral change correlates with the gross and histologic degree of thermal damage. Optical spectroscopy is a technology that allows real-time detection of thermal tissue damage. In this study, both F and Rd spectroscopy accurately defined the advancing hemorrhagic edge of the zone of ablation and the central coagulation zone. These results suggest that F and Rd spectroscopy can be used to create a real-time feedback system to accurately define RFA margins.

Mitochondrial calcium transport is regulated by P2Y1‐ and P2Y2‐like mitochondrial receptors

Ischemia‐reperfusion injury remains a major clinical problem in liver transplantation. One contributing factor is mitochondrial calcium (mCa2+) overload, which triggers apoptosis; calcium also regulates mitochondrial respiration and adenosine 5′‐triphosphate (ATP) production. Recently, we reported the presence of purinergic P2Y1‐ and P2Y2‐like receptor proteins in mitochondrial membranes. Herein, we present an evaluation of the functional characteristics of these receptors. In experiments with isolated mitochondria, specific P2Y1 and P2Y2 receptors ligands: 2‐methylthio‐adenosine 5′‐diphosphate (2meSADP) and uridine 5′‐triphosphate (UTP), respectively, were used, and mitochondrial calcium uptake was measured. 2meSADP and UTP had a maximum effect at concentrations in the range of the known P2Y1 and P2Y2 receptors. The P2Y inhibitor phosphate‐6‐azophenyl‐2′,4′‐disulfonate (PPADS) blocked the effects of both ligands. The phospholipase C (PLC) antagonist U73122 inhibited the effect of both ligands while its inactive analog U73343 had no effect. These data strongly support the hypothesis that mitochondrial Ca2+ uptake is regulated in part by adenine nucleotides via a P2Y‐like receptor mechanism that involves mitochondrial PLC activation. J. Cell. Biochem. 99: 1165–1174, 2006.

Reversed activity of mitochondrial adenine nucleotide translocator in ischemia-reperfusion

Background. Graft dysfunction as a result of preservation injury remains a major clinical problem in liver transplantation. This is related in part to accumulation of mitochondrial calcium. In an attempt to sustain cell and mitochondrial integrity during ischemia, intramitochondrial F0F1 adenosine triphosphate (ATP) synthase reverses its activity and hydrolyzes ATP to maintain the mitochondrial transmembrane potential (m&Dgr;&psgr;). It is not known how cytoplasmic ATP becomes available for hydrolysis by this enzyme. The authors hypothesized that mitochondrial adenine nucleotide translocator (ANT) reverses its activity during ischemia, making cytoplasmic ATP available for hydrolysis by F0F1 ATP synthase. Methods. Rat livers were perfused with cold University of Wisconsin solution at 4°C (39.2°F)through the portal vein and processed immediately or after 24 hr of cold storage. Mitochondria were separated by differential centrifugation. ATP-dependent mitochondrial calcium-45 (45Ca)2+ uptake was determined after incubation with ATP (5 mM) or adenosine diphosphate (ADP) (5 mM) with or without 15 &mgr;M of bongkrekic acid (BA), an ANT blocker; the nonhydrolyzable analog of ATP (adenosine 5′-&bgr;,&ggr;-imidotriphosphate [AMP-PNP]) served as the negative control. All measurements were performed in triplicate. Student t test, P <0.05 was taken as significant. Results. Inhibition of ANT by BA prevents mitochondrial Ca2+ accumulation in the presence of ATP and high 45Ca2+ concentrations, and increased extramitochondrial 45Ca2+ stimulated mitochondrial 45Ca2+ uptake in the presence of ATP but not ADP, AMP-PNP, or BA. Conclusions. These data demonstrate that ANT plays an important role in mitochondrial Ca2+ uptake under ischemic conditions by reversing its activity and allowing transport of extramitochondrial ATP into the matrix for hydrolysis by reversed F0F1 ATP synthase.

Inhibition of phospholipase C attenuates liver mitochondrial calcium overload following cold ischemia.

Background. Graft failure due to cold ischemia (CI) injury remains a significant problem during liver transplantation. During CI, the consumption of ATP and the increase in cellular Ca2+ concentration may result in mitochondrial Ca2+ (mCa2+) overload through the mCa2+ uniporter, which can ultimately lead to apoptosis and graft nonfunction. We recently identified phospholipase C-dl (PLC-dl) as a novel regulator of mCa2+ uptake in the liver, and now extend those studies to examine the role of mitochondrial PLC in liver CI injury. Methods. Rat livers were perfused with University of Wisconsin (UW) solution. Half was homogenized immediately; the other half was cold-stored for 24 hr in UW. Mitochondria were extracted by differential centrifugation and incubated in buffer containing ATP and 0.1 or 0.2 &mgr;M 45Ca2+. The selective PLC inhibitor, U-73122, was added to determine the effects of PLC inhibition on mCa2+ uptake following CI. Western blots and densitometry quantified mitochondrial PLC expression. Mito Tracker Red fluorescence microscopy was used to verify mitochondrial transmembrane potential. Results. Twenty-four hour CI caused a significant increase in mCa2+ uptake (P<0.001), and increasing extramitochondrial Ca2+ potentiated this effect. The PLC inhibitor, U-73122, decreased mCa2+ uptake in nonischemic mitochondria (P<0.001), and had a greater effect on CI mitochondria (P<0.001). Mitochondrial PLC-dl expression increased 175±75% following CI (P<0.05). Conclusions. These data demonstrate that PLC-dl is essential for mCa2+ uptake following CI, and that the PLC pathway may be sensitized by CI. The CI-induced increase in mitochondrial PLC-&dgr;1 expression represents a novel mechanism whereby mCa2+ uptake can increase independently of cytosolic conditions.

NFAT4 Deficiency Results in Incomplete Liver Regeneration Following Partial Hepatectomy

BACKGROUND Liver regeneration following partial hepatectomy requires the orchestration of highly regulated molecular pathways; a change in the abundance or activity of a specific gene product has the potential to adversely affect this process. The nuclear factor of activated T-cells (NFAT) transcription factors represent a family of gene transcription signaling intermediates that translate receptor-dependent signaling events into specific transcriptional responses using the Ras/Raf pathway. MATERIALS AND METHODS Eight-week old NFAT4 knockout (KO) mice and their wild type counterparts (Balb-c) underwent two-thirds partial hepatectomy. The animals were sacrificed and their livers were harvested at specific time points during regeneration. Recovery of liver mass was measured for each time point. PCR analysis was used to analyze expression levels of the immediate early genes c-fos, c-jun and c-myc as well as downstream effectors of NFAT4 including FGF-18 and BMP-4. RESULTS Hepatocyte proliferation and thus liver regeneration following hepatectomy was suppressed in NFAT4 knockout (KO) mice. Statistical significance was reached at 1 h, 7 d, and 10 d (P < 0.05) with a 22% median reduction in regeneration of liver mass in the NFAT4 KO mice by 10 d, at which time liver regeneration should be complete in mice. The immediate early gene c-fos was elevated in NFAT4 KO mice during early regeneration with a median value at 1 h and 1 d of 1.60E-08 and 1.09E-08 versus 6.10E-09 and 1.55E-09 in the Balb-c mice. C-jun, in contrast, was elevated during late regeneration in the NFAT4 KO mice (3.40E-09 and 5.67E-09 at 7 and 10 d, respectively) in comparison with the Balb-c mice (7.76E-10 and 1.24E-09, respectively.). NFAT2 was also up-regulated in the NFAT4 KO mice; however, no changes were detected in its downstream effectors, CCR1 and CCL3. CONCLUSIONS We demonstrated that NFAT4 deficiency impairs hepatic regeneration in a murine model proving that NFAT4 plays an important yet unclear role in liver regeneration; its absence may be compensated by c-fos, c-jun, and NFAT2 expression changes.