Chantal Éthier

Hepatic artery buffer response following left portal vein ligation: its role in liver tissue homeostasis

Occlusion of a lobar portal vein is known to induce atrophy of downstream liver lobes and hypertrophy of contralateral lobes. Changes in portal flow are known to be compensated by changes in hepatic arterial flow, thus defining the hepatic artery buffer response (HABR). To understand the role of liver flow in liver atrophy, we measured portal flow and hepatic artery flow after different degrees of left portal vein stenosis (LPVS). Surgery was performed to obtain 0, 43, 48, 59, 68, 72, 78, and 100% LPVS. Systemic and splanchnic blood flows were measured at 4 h or 7 days after surgery using radiolabeled microspheres. At 4 h, LPVS produced no changes in systemic hemodynamics. Increasing degrees of LPVS produced a significant decrease in left portal flow (P < 0.0001) and a fully compensatory increase in right portal flow (P < 0.0001) without significantly affecting total portal flow. Left hepatic artery flow increased by 210% (P = 0.002), and right hepatic artery flow decreased by 67% (P = 0.05) after full LPVS. There was a significant inverse correlation between portal and arterial flow changes induced by different degrees of LPVS in the left (r(2) = 0. 61) and right (r(2) = 0.41) lobes. Despite this HABR, we observed a reduction in left liver flow (-45%; P = 0.01) and an increase in right liver flow (+230%; P = 0.01) with 100% LPVS. At 7 days, a significant decrease in the weight of left liver lobes (-75%; P < 0. 0001) and a compensatory increase in the weight of the right lobes (+210%; P < 0.0001) were observed with 100% LPVS. Left and right liver flows were similar to results measured at 4 h, and HABR was still present. However, when expressed per gram of liver, liver flows were identical to results obtained with sham animals. Reduction in lobar portal flow is accompanied by an increase in ipsilateral hepatic artery flow and a compensatory increase in portal flow to the rest of the liver. In a given lobe, when compensatory HABR is overcome, liver weight changes occur so that at the end total liver flow per gram of liver tissue is restored. This suggests that in normal conditions liver flow is a major regulator of liver volume.Occlusion of a lobar portal vein is known to induce atrophy of downstream liver lobes and hypertrophy of contralateral lobes. Changes in portal flow are known to be compensated by changes in hepatic arterial flow, thus defining the hepatic artery buffer response (HABR). To understand the role of liver flow in liver atrophy, we measured portal flow and hepatic artery flow after different degrees of left portal vein stenosis (LPVS). Surgery was performed to obtain 0, 43, 48, 59, 68, 72, 78, and 100% LPVS. Systemic and splanchnic blood flows were measured at 4 h or 7 days after surgery using radiolabeled microspheres. At 4 h, LPVS produced no changes in systemic hemodynamics. Increasing degrees of LPVS produced a significant decrease in left portal flow ( P < 0.0001) and a fully compensatory increase in right portal flow ( P < 0.0001) without significantly affecting total portal flow. Left hepatic artery flow increased by 210% ( P = 0.002), and right hepatic artery flow decreased by 67% ( P = 0.05) after full LPVS. There was a significant inverse correlation between portal and arterial flow changes induced by different degrees of LPVS in the left ( r 2 = 0.61) and right ( r 2 = 0.41) lobes. Despite this HABR, we observed a reduction in left liver flow (-45%; P = 0.01) and an increase in right liver flow (+230%; P = 0.01) with 100% LPVS. At 7 days, a significant decrease in the weight of left liver lobes (-75%; P < 0.0001) and a compensatory increase in the weight of the right lobes (+210%; P < 0.0001) were observed with 100% LPVS. Left and right liver flows were similar to results measured at 4 h, and HABR was still present. However, when expressed per gram of liver, liver flows were identical to results obtained with sham animals. Reduction in lobar portal flow is accompanied by an increase in ipsilateral hepatic artery flow and a compensatory increase in portal flow to the rest of the liver. In a given lobe, when compensatory HABR is overcome, liver weight changes occur so that at the end total liver flow per gram of liver tissue is restored. This suggests that in normal conditions liver flow is a major regulator of liver volume.

Evaluation of hepatocyte injury following partial ligation of the left portal vein

BACKGROUND/AIMS Total ligation of the left portal vein is thought to induce both hepatocyte apoptosis and necrosis. The pathological impact of partial ligation of a branch of the portal vein has not yet been evaluated. METHODS We studied the degree of hepatocyte injury following 0, 43, 48, 59, 68, 72, 78 and 100% left portal vein stenosis in 200-g Sprague-Dawley male rats. Serum alanine aminotransferase levels, total body weight, and left and right liver lobe weights were measured at 2 and 7 days. Mitosis and 3H-thymidine labelling indices were measured as markers of proliferation; the apoptotic index and TUNEL stain were used as markers to measure apoptotic cell death. Necrosis was assessed morphologically. All these parameters were evaluated 2 days after ligation. RESULTS There was a direct relation between the increase in weight of the right lobes and the reduction in weight of the left lobes. The degree of weight change correlated significantly with the degree of stenosis. In the right lobes, mitosis and 3H-thymidine labelling were increased in proportion to the degree of stenosis. In the left lobes, the decrease in volume of hepatocytes correlated with the degree of ligation, especially in the pericentral areas. Necrosis was identified only when ligation was > or = 68%, this being associated with an increase in alanine aminotransferase levels. On the other hand, apoptotic cells were identified in increasing numbers, starting from the lowest degree of ligation to 100% ligation. This was found both morphologically and with TUNEL stain. CONCLUSIONS Partial ligation of the left portal vein induces left liver atrophy through hepatocyte volume loss and apoptosis. Necrosis is found only when the degree of ligation is severe.

Liver Fibrosis Protects Mice From Acute Hepatocellular Injury

BACKGROUND & AIMS Development of fibrosis is part of the pathophysiologic process of chronic liver disease. Although it is considered deleterious, it also represents a form of tissue repair. Deposition of extracellular matrix changes the cellular environment of the liver; we investigated whether it increases resistance to noxious stimuli and the role of changes in intracellular signaling to hepatocytes in mediating this effect. METHODS Primary cultures of mouse hepatocytes were exposed to type I collagen (COL1); cell injury was assessed by morphologic and biochemical criteria. The expression of Bcl-2 family members was evaluated by immunoblot analyses. Activation of extracellular signal-regulated kinase (ERK) was assessed using phospho-specific antibodies. Liver fibrosis was induced by repeated administration of thioacetamide or carbon tetrachloride to mice; mice were then exposed to Fas antibodies. RESULTS Hepatocytes exposed to COL1 were more resistant to a variety of hepatotoxins, in a dose-dependent manner, and had lower levels of Bad, Bid, and Bax proapoptotic proteins compared with control hepatocytes. Activation of ERK1/2 was stronger and quicker in hepatocytes exposed to COL1. The MEK1/2 inhibitors U0126 and PD98059 reversed the protective effects of COL1 and the decrease in proapoptotic proteins. Hepatocytes isolated from ERK1(-/-) mice were insensitive to the protective effect of COL1. Fibrotic livers from wild-type mice had high levels of phospho-ERK1 and were resistant to Fas-induced cell death. ERK1(-/-) mice lost this effect. CONCLUSIONS Production of COL1 during liver fibrosis induces a hepatoprotective response that is mediated by activation of ERK1 signaling.

EGF mediates protection against Fas-induced apoptosis by depleting and oxidizing intracellular GSH stocks.

Several pieces of evidence have demonstrated the importance of reduction/oxidation (redox) signaling in biological processes, including sensitivity toward apoptosis. In parallel, it was recently reported that growth factors induce the generation of reactive oxygen species (ROS). Therefore, we tested the hypothesis that the anti‐apoptotic effect of epidermal growth factor (EGF) was mediated by changes in the redox state of hepatocytes through changes in GSH stocks. Isolated mouse hepatocytes were cultured and exposed to anti‐Fas stimulation in order to induce apoptosis. Cell death by apoptosis was assessed by Hoechst 33258 staining and by measuring caspase‐3 proteolysis activity. Cell treatment with EGF significantly decreased total (GSx) and reduced (GSH) glutathione levels in the presence and the absence of anti‐Fas. Furthermore, glutathione reductase activity was lower in EGF‐treated cultures (by 28%) as compared to untreated cultures which lead to a significant decline in GSH/GSx ratio. These effects were found to be EGF‐receptor tyrosine kinase activity dependent. Co‐stimulation of cells with anti‐Fas and EGF attenuated caspase‐3 activation and cell death by apoptosis by 70%. GSH monoethylester (GSHmee) significantly attenuated the effect of EGF on GSH and GSH/GSx ratio. It caused an increase in caspase‐3 activation and in the percentage of apoptotic cells in anti‐Fas + EGF‐treated cells, thus resulting in a 53% decline in the protective effect of EGF. In conclusion, EGF induces a significant and specific depletion and oxidization of intracellular GSH, paralleled by a protection against Fas‐induced apoptosis. GSH replenishment partly counteracted these effects suggesting that GSH depletion contributed to the protective effect of EGF against caspase‐3 activation and cell death by apoptosis. J. Cell. Physiol. 198: 62–72, 2004.

Hepatic expression of regeneration marker genes following partial hepatectomy in the rat: Influence of 1,25-dihydroxyvitamin D3 in hypocalcemia

BACKGROUND/AIMS Vitamin D (D) depletion is a common feature of chronic liver diseases. In past years, disturbances in calcium metabolism involving inadequate D and parathyroid hormone status have been reported to significantly impair the hepatic regeneration process following partial hepatectomy in the rat. The purpose of this study was to investigate how hypocalcemia and D deficiency affect specific cell markers of hepatic compensatory growth. METHODS Steady-state mRNA levels of gene markers of the regeneration process were investigated following 2/3 partial hepatectomy. The response of hypocalcemic D-depleted rats was compared to that of animals whose calcium status had been normalized by repletion with the active D hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). RESULTS The transcript for the major hepatic mitogen HGF increased in both groups after partial liver resection but the increase was significantly lower as well as delayed in livers obtained from calcium deficient rats in the prereplicative phase of the regeneration process. TGF alpha mRNA levels were also found to be significantly lower in calcium deficient rats at all time-points following partial hepatectomy, while the relative behavior of the tandem TGF alpha-EGFR indicated an early dominant effect in normocalcemic 1,25(OH)2D3-repleted animals. HGF-c-met mRNA levels also indicated that the 1,25(OH)2D3-repleted animals reacted more promptly to the regeneration stimuli. Indeed, while relative (1,25(OH)2D3/D-Ca- ratio) maximum mRNA levels were observed 12 h following liver resection in 1,25(OH)2D3-treated animals, relative peak levels were only apparent 24 h post-surgery in hypocalcemic rats. Maximum cyclin D1 (a marker of the G1 phase of the cell cycle) mRNA occurred between 8-18 h after partial hepatectomy in 1,25(OH)2D3-repleted animals to return to base-line value thereafter, but in hypocalcemic rats the transcript levels remained significantly below 1,25(OH)2D3-repleted animals during the prereplicative period with increases above initial values between 12-24 h post-surgery. Both cyclin A (an S phase marker) transcripts (1.8 and 2.9 kb) were influenced by the regeneration process. The transcripts significantly and sharply increased in hypocalcemia between 30-36 h following partial hepatectomy to decrease thereafter, while the increase was observed between 24-30 h, and at 48 h (1.8 kb) in 1,25(OH)2D3-repleted animals. Liver weight recovery was also found to be decreased in D-depleted rats over the 48 h period of observation. CONCLUSIONS Our data further confirm the presence of an impaired regeneration process in hypocalcemia of D deficiency which seems to be associated with gene markers indicating an inefficient transit across the G1 phase of the cell cycle.

Hypocalcemia modifies the intra-cellular calcium response to the α1-adrenergic agent phenylephrine in rat hepatocytes

In vivo, extracellular calcium ([Ca2+]e) homeostasis is maintained within a very narrow range by the calcium regulating hormones. At the cellular level, the response to many agents is transduced by changes in cytosolic Ca2+ ([Ca2+]i) which involves both mobilization of cellular pools and entry of [Ca2+]e through plasma membrane channels. To investigate the cellular effects of chronic hypocalcemia (Ca-) on [Ca2+]i homeostasis, hepatocytes, a cell type well characterized for its [Ca2+]i response, were used. Data indicate that Ca- leads to a significant shift to the left in the basal resting cytosolic Ca2+ concentration distribution curve with half-maximum cumulative frequency of 119 versus 149 nM in Ca- and normal rats (N) respectively (P < 0.0001). The response to the alpha 1-adrenergic agonist phenylephrine (Phe) was also influenced by Ca- with a dampening of the dose-response curve, a significant decrease in the frequency of sustained responses (P < 0.001), and significant changes in the oscillation pattern. Indeed, hepatocytes obtained from Ca- exhibited a higher frequency of large amplitude, low frequency oscillations than N most particularly at the 2 and 5 microM Phe dose while N predominantly exhibited low amplitude, high frequency oscillations on sustained plateaus (P < 0.001). IP3 receptor (IP3R) binding studies and Ca2+ mobilization from IP3-sensitive pools showed that IP3R was highly sensitive to the prevailing Ca2+ with, in the range of resting [Ca2+]i, R affinity significantly lower in Ca- than in N. Upon exposure of permeabilized cells to 25 microM IP3, Ca2+ mobilization from the IP3-sensitive intracellular pool was significantly reduced by Ca- (P < 0.05) suggesting a decrease in the IP3-mobilizable Ca2+ pool in Ca-. Our results indicate that hypocalcemia significantly alters [Ca2+]i signalling by perturbing the initial response to agonist and the [Ca2+]i response pattern. In addition, the decrease in Ca2+ mobilization from IP3-sensitive pools suggests that hypocalcemia may also lead to a decrease in the Ca2+ content of intracellular pools.

Primary cultures of human hepatocytes isolated from hepatitis C virus-infected cirrhotic livers as a model to study hepatitis C infection

Background/Aim: Since the discovery of hepatitis C virus (HCV), researchers have encountered difficulties with in vitro models. The aim of this study was to determine whether HCV‐infected human primary hepatocytes, isolated from cirrhotic livers at liver transplantation, can be used as a model to study HCV infection.

Anti-apoptotic effect of insulin on normal hepatocytes in vitro and in vivo.

Liver growth factors are known to be anti-apoptotic for hepatocytes. The potential of insulin, a liver co-mitogen, has not been thoroughly evaluated. We studied the anti-apoptotic role of insulin on primary cultures of rat hepatocytes exposed to transforming growth factor-β (TGF-β) as the apoptotic agent and in the left portal vein ligation model (LVPL) of liver atrophy. Results show that insulin decreases apoptosis of TGF-β-treated hepatocyte cultures by 43% (P < 0.002) and the alanine amino transferase (ALT) release by 49% (P < 0.001). Left lobes of LPVL animals displayed a significant increase in the levels of TGF-β mRNA. In LPVL rats receiving continuous infusion of insulin in the left lobes, the weight of the atrophic lobes was higher over a 7-day period in comparison to control animals. This was associated with lower levels of serum ALT and with a five-fold decrease in the apoptotic index in the left lobes (P < 0.0001). Induction of Akt phosphorylation and increased expression of Bcl-xl were observed in the left lobes of insulin-treated animals. In conclusion, these results show that insulin is anti-apoptotic for normal hepatocytes both in vitro and in vivo and that the action of insulin is associated with increased Bcl-xl expression and Akt activation.

Influence of the calcium and vitamin D endocrine system on the "priming" of the liver for compensatory growth.

The liver is known to be sensitive to dietary challenge and to reduction in its functional mass. To investigate the influence of the vitamin D endocrine system on the hepatic growth response, liver DNA synthesis (S phase of cell cycle) was primed by dietary manipulation (high carbohydrate/protein-free diet x 3 d followed by high protein diet x 15-18 hr) in animals presenting various calcium and vitamin D status. Data indicate the dietary manipulation increased [3H]thymidine incorporation into DNA following vitamin D3 (D3) or 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) administration (p less than 0.0001) while vitamin D depleted (D-) rats (both hypo and normocalcemic) showed no significant increases over unchallenged rats (p less than 0.0001). Studies on the hepatic EGF receptor indicate that while no significant between-group difference was observed in receptor density or affinity, evaluation of the receptor density in relation to the [3H]thymidine incorporation response revealed a higher receptor density in responders (D3 and 1,25(OH)2D3 supplemented groups) with 30.2 +/- 1.4% maximum binding than in non-responders (hypo and normocalcemic D- groups) with 25.4 +/- 1.8% maximum EGF binding (p less than 0.03); EGF EC50 was found to be 50.2 +/- 4.4 and 63.8 +/- 9.7 ng/ml in responders and non-responders respectively (p = 0.1). These data indicate that vitamin D depletion is accompanied by hyporesponsiveness when challenged by a dietary protocol known to prime the liver for growth inducing stimuli.