Effect of caloric restriction on hepatic sinusoidal system and stellate cells in mice.

Aging associated changes in liver include reduced hepatic blood flow, increased number of stellate cells, and collagen deposits in perisinusoidal space. We tested the possibility of mitigating these changes with caloric restriction. Two-month-old mice were subjected to 30 percent caloric restriction for 12 months and then examined for the effect of caloric restriction on the sinusoidal network, collagen deposition, and the number of stellate cells. Using intravital fluorescence microscopy, assessments were made on sinusoidal diameter, density, volumetric flow, perfusion index, and autofluorescence of vitamin A that was primarily stored with lipid droplets in stellate cells. A significant effect was observed in the vitamin A autofluorescence of stellate cells; stellate cell associated fluorescence was diminished in terms of number and size of fluorescent spots. Caloric restriction reduced collagen deposits in liver sections and lowered the gene expression of α 1-(I) collagen but not α -smooth muscle actin. No differences were detected in sinusoidal dimension measurements. Our results showed that caloric restriction was effective in ameliorating the increase in stellate cells and the mild fibrosis in old mice. However, caloric restriction had no impact on stellate cell activity level as indicated by the unaffected α -smooth muscle actin expression.

Altered endothelin-1 signaling in production of thromboxane A2 in kupffer cells from bile duct ligated rats.

Kupffer cells (KCs), the liver resident macrophages accounting for 80-90% of the total population of fixed tissue macrophages in the body, not only play a key role in host defense via removing particulate materials from the portal circulation, but may also contribute to the pathogenesis of various liver diseases. We have previously demonstrated that KCs play an important role in controlling portal hypertension and hepatocellular injury via releasing thromboxane A2 (TXA2) in early fibrosis induced by one-week bile duct ligation (BDL). Production of TXA2 is controlled by cytosolic phospholipase A2 (cPLA2) that is activated by the interaction of entothelin-1 (ET-1) with its G-protein coupled ET receptor B (ETBR). However, the signaling pathways that contribute to the ET-1-induced activation of cPLA2 and production of TXA2 in KCs in the normal healthy or injured livers are not yet clear, which are investigated in the present study using isolated KCs from one-week BDL or sham rats. The pharmacological inhibition of cPLA2 or chelation of intracellular calcium abrogated the ET-1 induction of TXA2 from KCs. Compared to those from sham rats, KCs from BDL animals displayed a significantly enhanced responsiveness of p38 MAPK to ET-1, increased ETBR and Galphai subunit but decreased Galphaq and Galpha11 expression. Inhibition of ERK1/2 or Gq signaling abrogated significantly the ET-1 induction of TXA2 in sham KCs but only slightly in BDL KCs. In contrast, inhibition of p38 MAPK and Gi signaling markedly attenuated the ET-1 induction of TXA2 in BDL KCs but had no effect in sham KCs. Lastly, inhibition of PLC or PKC abrogated ET-1 induction of TXA2 in KCs from both sham and BDL groups. The hepatic stress (such as BDL) induces significant modifications in the receptor and intermediates of ET-1 signaling in KC and subsequently alters ET-1 signaling mechanisms, particularly a shift from Gq induced signaling to Gi induced signaling, in the activation of cPLA2 and production of TXA2 in response to ET-1.

Improved preservation of warm ischemic livers by hypothermic machine perfusion with supplemented University of Wisconsin solution.

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

Inhibition of TXA synthesis with OKY-046 improves liver preservation by prolonged hypothermic machine perfusion in rats.

BACKGROUND AND AIM: We previously reported that hypothermic machine perfusion (HMP) for liver preservation is feasible, but hepatic microcirculatory dysfunction and significant liver damage remain major obstacles in its application when the preservation is extended to 24 h. The underlying injury mechanism is not well understood. The present study sought to investigate the role of thromboxane A(2) (TXA(2)) in the pathogenesis of liver injury after prolonged HMP. METHODS: Livers isolated from Sprague-Dawley rats were subjected to continuous machine perfusion with University of Wisconsin (UW) solution at a flow rate of 0.4 mL/min/g liver at 4 degrees C for 24 h. A specific TXA(2) synthase inhibitor, OKY-046 (OKY), was added to UW solution during the preservation period and to the Krebs-Henseleit buffer during reperfusion. The performance of the livers after preservation was evaluated using an isolated liver perfusion system with Krebs-Henseleit buffer at a flow rate of 15 mL/min at 37 degrees C for 30 min. RESULTS: Prolonged HMP induced a significant release of TXA(2) into the portal circulation as indicated by markedly increased levels of TXB(2) in the perfusate during reperfusion (at 30 min, 1447.4 +/- 163.6 pg/mL vs 50.91 +/- 6.7 pg/mL for control). Inhibition of TXA(2) synthesis with OKY significantly decreased releases of TXA(2) (69.8 +/- 13.4 pg/mL) concomitant with reduced lactate dehydrogenase (LDH) releases (at 30 min, HMP + OKY: 144.9 +/- 27.9 U/L; HMP: 369.3 +/- 68.5 U/L; simple cold storage or SCS: 884.4 +/- 80.3 U/L), decreased liver wet/dry weight ratio (HMP + OKY vs SCS and HMP: 3.6 +/- 0.3 vs 4.4 +/- 0.1 and 3.9 +/- 0.2, respectively) and increased hyaluronic acid uptake (at 30 min, HMP + OKY vs SCS, HMP: 33.1 +/- 2.9% vs 13.9 +/- 3.6%, 18.6 +/- 2.4%, respectively). Liver histology also showed significant improvement in tissue edema and hepatocellular necrosis with OKY compared with HMP without OKY. CONCLUSION: The results demonstrate that TXA(2) is involved in the development of hepatocellular injury induced by HMP, and inhibition of TXA(2) synthesis during preservation and reperfusion protects liver hepatocytes and sinusoidal endothelial cells from injuries caused by prolonged HMP.

LPS exacerbates endothelin-1 induced activation of cytosolic phospholipase A2 and thromboxane A2 production from Kupffer cells of the prefibrotic rat liver.

BACKGROUND/AIMS: Thromboxane A2 (TXA2) has been suggested to play a significant role in the development of portal hypertension in fibrosis, and Kupffer cell (KC) derived TXA2 has been shown to mediate the hyperresponsiveness of the portal circulation to the vasoconstrictive actions of endothelin-1 (ET-1) during endotoxemia. The aim of this study was to determine whether the double stresses of prefibrotic changes and endotoxemia additively activate KC to increase release of TXA2 in response to ET-1, resulting in elevated portal resistance. METHODS: One week Bile duct ligation (BDL) rats and sham-operated controls were subjected to isolated liver perfusions following LPS or saline for 6h. In a separate experiment, KC were isolated from BDL or sham rats and incubated with LPS or saline for 6h before the ET-1 treatment. RESULTS: The double stresses of early fibrosis and LPS resulted in a greater sustained increase in portal pressure in response to ET-1 in BDL rats, and this increase correlated well with the much enhanced release of TXA2 in the perfusate. Media from the cultured KC showed significantly greater TXA2 release in response to ET-1 in BDL group than those in sham group, and LPS exacerbated this effect. Protein levels of cytosolic phospholipase A2 (cPLA2), cyclooxygenase-2, and thromboxane synthase were also significantly elevated in KC from BDL rats. ET-1 produced a marked increase in cPLA2 activation as measured by the phosphorylation of cPLA2 in KC of both BDL and sham groups. LPS greatly exacerbated the activation of cPLA2. CONCLUSIONS: The data suggest that the double stresses additively activate KC with an upregulation of the key enzymes in the TXA2 biosynthesis and release increased amount of TXA2 via the augmented activation of cPLA2 in response to ET-1, which leads to the increased portal resistance and ultimately hepatic microcirculatory dysfunction.

Hepatic arterial flow becomes the primary supply of sinusoids following partial portal vein ligation in rats.

BACKGROUND AND AIM: Partial portal vein ligation (PPVL) is a commonly used procedure to induce prehepatic portal hypertension in animal models. The aim of this study was to test the hypothesis that the hepatic arterial flow becomes the primary source feeding the sinusoids in the liver after PPVL. METHODS: Sprague-Dawley rats underwent either sham operation or partial portal vein ligation (PPVL). The number of vessels in the liver at 2 weeks postoperatively was determined by factor VIII immunolocalization and the gene expression of angiogenic factors was assessed by RT-PCR. The total hepatic arterial supply to the liver was measured using the fluorescent microsphere injection technique. To further test the hypothesis, two additional groups of rats underwent hepatic artery ligation (HAL) or PPVL plus HAL (PPHAL). The integrity of hepatic microcirculation was then evaluated in all four groups by intravital microscopy. RESULTS: At 2 weeks after operation, the number of vessels detected by factor VIII staining was significantly higher in PPVL compared to sham. Densitometric analysis of RT-PCR bands revealed a significant increase of vascular endothelial growth factor gene expression in PPVL compared to sham. Arterial flow to the liver measured by fluorescent microspheres was increased by 190% in PPVL compared to sham. When all four groups were compared, no prominent histological abnormality was observed in sham, HAL, and PPVL groups; however, PPHAL livers showed focal necrosis and inflammatory cell infiltration around the portal triads. Additionally, only the PPHAL livers showed a decreased sinusoidal diameter and significantly lower perfusion index (PPHAL 42.9+/-6.1; sham 85.7+/-7.0, PPVL 80.2+/-6.5, HAL 70.9+/-4.5). CONCLUSIONS: These results suggest that the hepatic artery flow becomes the primary source for the blood supply of sinusoids and the compensatory change in the hepatic arterial system plays a critical role in maintaining microcirculatory perfusion following the restriction of the portal vein flow by PPVL.

Inhibition of endothelin-1-mediated up-regulation of iNOS by bosentan ameliorates endotoxin-induced liver injury in cirrhosis.

Endothelin-1 (ET-1) has been shown to regulate the expression of various genes in addition to its vasoconstrictor role in the liver. Elevated levels of ET-1 during cirrhosis play an important role in the observed microcirculatory dysfunction; however, its role as a transcription regulator remains unclear. This study aimed to determine the role of ET-1 in the hepatic gene expression of vasomediators after cirrhosis in response to LPS. Cirrhosis was induced by bile duct ligation (BDL) for 1 or 3 weeks in male Sprague-Dawley rats. Following 1 or 3 weeks of BDL or sham operation (sham), rats received an intravenous (i.v.) injection of bosentan, a dual-selective ETA/B receptor antagonist (30 mg/kg bw) or saline, and an intraperitoneal (i.p.) injection of LPS (1 mg/kg bw). Plasma alanine aminotransferase (ALT) levels were significantly elevated in 1- and 3-week BDL animals. Six hours following LPS, the elevated ALT levels were markedly exacerbated in 3-week BDL animals, which were significantly ameliorated with bosentan treatment. LPS resulted in increased ET-1, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 mRNA expressions in both sham and BDL rats. Bosentan significantly inhibited the up-regulations of ET-1, iNOS, and COX-2 mRNA. Our data strongly suggest that ET-1 plays an important role in up-regulating the expression of iNOS, COX-2, and ET-1 itself in hepatic tissue following LPS challenge, which may contribute to the observed hepatocellular injury during endotoxemia in cirrhosis. Thus, due to significant increases in ET-1 levels during cirrhosis, ET-1 receptor blockade may prove to be of great therapeutic value in the treatment of cirrhotic patients exposed to secondary injuries such as endotoxemia.

Thromboxane A2 from Kupffer cells contributes to the hyperresponsiveness of hepatic portal circulation to endothelin-1 in endotoxemic rats.

We examined the role of thromboxane A2 (TXA2) in LPS-induced hyperresponsiveness of hepatic portal circulation to endothelins (ETs) and whether Kupffer cells are the primary source of TXA2 release in response to ET-1 in endotoxemia. After 6 h of LPS (1 mg/kg body wt ip) or saline (control), liver was isolated and perfused with recirculating Krebs-Henseleit bicarbonate buffer at a constant flow rate (100 ml.min(-1).kg body wt(-1)). ET-1 (10 pmol/min) was infused for 10 min. Portal pressure (PP) was continuously monitored during perfusion. Perfusate was sampled for enzyme immunoassay of thromboxane B2 (TXB2; the stable metabolite of TXA2) and lactate dehydrogenase (LDH) assay. ET-1 infusion resulted in a significantly greater increase of PP in the LPS group than in controls. Both TXA2 synthase inhibitor furegrelate (Fureg) and TXA2 receptor antagonist SQ-29548 (SQ) substantially blocked enhanced increase of PP in the LPS group (4.9 +/- 0.4 vs. 3.6 +/- 0.5 vs. 2.6 +/- 0.6 mmHg for LPS alone, LPS + Fureg, and LPS + SQ, respectively; P < 0.05) while having no significant effect on controls. GdCl3 for inhibition of Kupffer cells had similar effects (4.9 +/- 0.4 mmHg vs. 2.9 +/- 0.4 mmHg for LPS alone and GdCl3 + LPS, respectively; P < 0.05). In addition, the attenuated PP after ET-1 was found concomitantly with significantly decreased releases of TXB2 and LDH in LPS rats treated with Fureg, SQ, and GdCl3 (886.6 +/- 73.4 vs. 110.8 +/- 0.8 vs. 114.8 +/- 54.7 vs. 135.2 +/- 45.2 pg/ml, respectively; P < 0.05). After 6 h of LPS, Kupffer cells in isolated cell preparations released a significant amount of TXA2 in response to ET-1. These results clearly indicate that hyperresponsiveness of hepatic portal circulation to ET-1 in endotoxemia is mediated at least in part by TXA2-induced receptor activation, and Kupffer cells are likely the primary source of increased TXA2 release.

Remote trauma sensitizes hepatic microcirculation to endothelin via caveolin inhibition of eNOS activity.

This study addresses the microvascular mechanisms by which a remote, mild stress such as blunt trauma sensitizes the liver to injury. Rats received closed femur fracture (FFx), and 24 h later livers were isolated and perfused at a similar starting flow rate for assessment of vascular response to endothelin-1 (ET-1). Sinusoidal volumetric flow (QS), red blood cell velocity (VRBC), and sinusoidal diameter (Ds) were determined by intravital microscopy. Baseline portal resistance in livers from FFx rats was not changed. The FFx group showed a lower baseline VRBC (322.9 +/- 26.4 and 207.3 +/- 17.2 microm/s in sham and FFx,) and QS (28.4 +/- 4.2 and 17.6 +/- 2.1 pL/s in sham and FFx, P < 0.05). ET-1 caused a decrease in the VRBC in sham but no change after FFx. In contrast, Ds was unchanged by ET-1 in sham but decreased in FFx (10.3 +/- 0.4 to 10.7 +/- 0.5 vs. 10.6 +/- 0.4 to 9.0 +/- 0.4 microm at 10 min in sham and FFx groups, P < 0.05). The overall result of these changes was a greater decrease in sinusoidal flow in FFx compared with sham. There was no significant change in mRNA for ET-1, endothelin A (ETA) receptor, or iNOS (inducible nitric oxide synthase) in FFx compared with sham. However, endothelin B (ETB) receptor mRNA and eNOS (endothelial nitric oxide synthase) mRNA were increased in the FFx group (ETB, 54.81 +/- 8.08 in sham vs. 83.28 +/- 8.19 in FFx; eNOS, 56.11 +/- 2.53 in sham vs. 83.31 +/- 5.51 in FFx; P < 0.05) while the levels of these proteins remained unchanged. Caveolin-1 (cav-1) protein levels were elevated in FFx, and coimmunoprecipitation with both ETB and eNOS showed increased associations with these proteins, suggesting a possible inactivation of eNOS. The eNOS activity was also blunted in FFx animals in the presence of increased cav-1 expression. Taken together, these results demonstrate that remote trauma sensitizes the liver to the sinusoidal constrictor effect of ET-1. We propose that this hyperresponsiveness occurs as a result of uncoupling of the ETB receptor from eNOS activity mediated by interaction of eNOS and possibly the ETB receptor with increased caveolin-1. This vascular sensitization that occurs after FFx may contribute to the exacerbation of injury during subsequent stresses.

Pronlonged hypothermic machine perfusion preserves hepatocellular function but potentiates endothelial cell dysfunction in rat livers.

BACKGROUND: Although hypothermic machine perfusion (HMP) preservation has been shown to improve organ function and to expand the organ donor pool, problems still exist with the current HMP technology for liver preservation. The present study was conducted to investigate endothelial and hepatocellular functions following extended HMP (> r =24 hr) in rat liver model. METHODS: Following 24-hour hypothermic HMP with University of Wisconsin (UW) solution or 24-hour simple cold storage (SCS), livers were reperfused with Krebs-Henseleit buffer solution at 37 degree C for 30 minutes. Hepatocyte damage and function were assessed by measuring lactate dehydrogenase (LDH) activity, bile production, and indocyanine green (ICG) extraction. Sinusoidal endothelial cell (SEC) function and permeability were determined by hyaluronic acid (HA) uptake and multiple indicator dilution (MID) method, respectively. RESULTS: After 24-hour hypothermic preservation, HMP livers showed lower released LDH levels, higher bile flow rate, and greater hepatic ICG uptake compared with SCS livers. However, LDH levels became significantly higher in HMP than in SCS after 30 minutes of warm perfusion. The increased enzyme levels were accompanied by a significant increase in endothelial permeability to albumin and a decrease in hyaluronic acid uptake in HMP compared to SCS. Liver wet/dry weight ratio confirmed a greater edema in HMP livers than SCS livers. CONCLUSION: These results suggest that 24-hour hypothermic HMP may help preservation of hepatocyte function, but endothelial cell dysfunction during the cold preservation may play a key role in hepatocyte dysfunction and parenchymal cell death upon reperfusion.

Ex-vivo study of flow dynamics and endothelial cell structure during extended hypothermic machine perfusion preservation of livers.

Liver transplantation is often the only effective treatment for end stage liver diseases resulting from cirrhosis, hepatitis, progressive jaundice, and biliary atresia. Hypothermic machine perfusion (HMP) preservation may enhance donor pool by extending preservation time and reclaiming marginal donor livers including those from non-heart beating donors (NHBD), as demonstrated in the kidney. However, current HMP protocols have not been successful in improving extended preservation of livers and the major cause of preservation injury remains unknown. An intravital microscopy study was conducted to understand the flow dynamics of sinusoidal perfusion during 24h HMP with cold modified University of Wisconsin (UW) solution. Fluorescein isothiocynate (FITC) labeled albumin was utilized to visualize microvascular space and FITC labeled red blood cells (RBCs) were used to visualize flow dynamics during HMP. A heterogeneous flow pattern with regions of red cell stasis was observed after 24-h HMP. To examine the cause of red cell stasis, intravital and confocal microscopy studies of endothelial cells (ECs) structure labeled with DiI acetylated low-density lipoprotein (DiI acLDL) were conducted. These studies suggest that morphological changes in EC structures occurred during 24h HMP, which may cause obstruction to the sinusoidal flow. Histological findings confirm these results. As a result, heterogeneous flow pattern, red cell stasis, and edema occur, which may lead to the failure of these tissues following extended HMP.

Survival transplantation of preserved non-heart-beating donor rat livers: preservation by hypothermic machine perfusion.

BACKGROUND: Non-heart-beating donor (NHBD) livers are an untapped source with the potential to provide relief to the current donor shortage problem. Hypothermic machine perfusion (MP) has the potential to reclaim and preserve these marginal donor organs. METHODS: This study compared 5-day survival in a rat NHBD liver transplantation model with simple cold storage (SCS) and MP-preserved tissues that had experienced 30 min of warm ischemia followed by a 5-hr preservation period with the University of Wisconsin solution. Total release of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT) were determined at major time points. Bilirubin levels and histology were examined after 5-day survival. RESULTS: Six of seven control livers and five of six MP livers survived, whereas SCS tissues had survival in zero of seven. The results showed that MP livers had reduced release of LDH and ALT after 5 hr of storage, 5.07+/-1.42 and 2.02+/-0.69 U (mean+/-SE), respectively, compared with SCS, 15.54+/-0.81 and 3.41.3+/-0.73 U, respectively. Bilirubin values after 5-day survival of MP livers (1.17+/-0.49 mg/dL) were comparable to controls (0.91+/-0.36 mg/dL). Histology confirms that SCS displayed increased necrosis and MP tissue showed regions of near normal hepatic structure. CONCLUSIONS: These results suggest that MP for 5 hr improves survival and reduces cellular damage of liver tissue that has experienced 30 min of warm ischemia when compared with SCS tissues. Further studies need to be conducted, but this study suggests that MP preservation has the potential to reclaim and preserve NHBD liver tissues.

Role of thromboxane A2 in early BDL-induced portal hypertension.

Although the mechanisms of cirrhosis-induced portal hypertension have been studied extensively, the role of thromboxane A(2) (TXA(2)) in the development of portal hypertension has never been explicitly explored. In the present study, we sought to determine the role of TXA(2) in bile duct ligation (BDL)-induced portal hypertension in Sprague-Dawley rats. After 1 wk of BDL or sham operation, the liver was isolated and perfused with Krebs-Henseleit bicarbonate buffer at a constant flow rate. After 30 min of nonrecirculating perfusion, the buffer was recirculated in a total volume of 100 ml. The perfusate was sampled for the enzyme immunoassay of thromboxane B(2) (TXB(2)), the stable metabolite of TXA(2). Although recirculation of the buffer caused no significant change in sham-operated rats, it resulted in a marked increase in portal pressure in BDL rats. The increase in portal pressure was found concomitantly with a significant increase of TXB(2) in the perfusate (sham vs. BDL after 30 min of recirculating perfusion: 1,420 +/- 803 vs. 10,210 +/- 2,950 pg/ml; P < 0.05). Perfusion with a buffer containing indomethacin or gadolinium chloride for inhibition of cyclooxygenase (COX) or Kupffer cells, respectively, substantially blocked the recirculation-induced increases in both portal pressure and TXB(2) release in BDL group. Hepatic detection of COX gene expression by RT-PCR revealed that COX-2 but not COX-1 was upregulated following BDL, and this upregulation was confirmed at the protein level by Western blot analysis. In conclusion, these results clearly demonstrate that increased hepatic TXA(2) release into the portal circulation contributes to the increased portal resistance in BDL-induced liver injury, suggesting a role of TXA(2) in liver fibrosis-induced portal hypertension. Furthermore, the Kupffer cell is likely the source of increased TXA(2), which is associated with upregulation of the COX-2 enzyme.

Potentiated hepatic microcirculatory response to endothelin-1 during polymicrobial sepsis.

We conducted this study to elucidate the role of endothelins (ET-1) in mediating the hepatic microcirculatory dysfunction observed in response to sepsis. Following 24 h of cecal ligation and puncture (CLP), we performed intravital microscopy both in vivo and on isolated perfused livers. Portal resistance increased in response to ET-1 in both sham and septic rats, with no significant difference between the two in either in vivo or in isolated livers. Sinusoidal volumetric flow (Qs) was evaluated using red blood cell velocity (V(RBC)) and sinusoidal diameter (Ds) to determine microvascular hemodynamic integrity. Qs decreased in response to ET-1 in livers from CLP rats compared with sham (P < 0.05, CLP vs. sham) in both in vivo and isolated livers. In vivo infusion of ET-1 resulted in greater constriction of sinusoids in the CLP group compared with sham (P < 0.05), resulting in higher sinusoidal resistance. Microvascular hyper-responsiveness was accompanied by hepatocellular injury in CLP rats, but not in sham rats. RT-PCR was performed to measure mRNA levels of ET-1, its receptors ET(A) and ET(B), inducible and constitutive nitric oxide (NO) synthase (iNOS and eNOS, respectively), and heme oxygenase 1 (HO-1). After CLP, both ET-1 and ET(B) mRNA increased, whereas ET(A) mRNA tended to decrease, although the change was not statistically significant. Livers from CLP rats showed no significant change in levels of eNOS mRNA, but showed a significant increase in iNOS expression (13.5-fold over sham). There was no change in the level of HO-1 mRNA between sham and CLP groups. Taken together, these results suggest that sepsis sensitizes the hepatic microcirculation to ET-1. More importantly, an impaired microcirculatory flow due to ET-1 in sepsis contributes to hepatic injury. Further, localized imbalances between endothelins and NO may mediate the altered microvascular response during sepsis.

Endothelin-1 and heme oxygenase-1 as modulators of sinusoidal tone in the stress-exposed rat liver.

Heme oxygenase (HO)-1 is up-regulated after ischemia/reperfusion and contributes to maintenance of hepatic perfusion and integrity. Blockade of HO-1 leads to an increased portal pressor response in the stress-exposed liver. We tested whether the increase in portal pressure reflects unmasking of a concomitant up-regulation of the vasoconstrictor endothelin (ET)-1. Hemorrhagic shock induced messenger RNAs encoding HO-1 (16-fold) and ET-1 (9-fold) with a similar time course in the liver. At maximum induction of both mediators, rats received either vehicle or the endothelin ET(A/B) antagonist bosentan (10 mg/kg intravenously). Subsequently, the HO pathway was blocked in all animals by tin-protoporphyrin (SnPP)-IX (50 micromol/kg intravenously). Portal and sinusoidal hemodynamics were measured using microflow probes and intravital microscopy, respectively. Blockade of the HO pathway led to a significant increase in portal resistance (sham/SnPP-IX, 0.17 +/- 0.046 mm Hg. min. mL(-1); shock/vehicle/SnPP-IX, 0.57 +/- 0.148 mm Hg. min. mL(-1); P <.05) and a decrease in sinusoids conducting flow (shock/vehicle/SnPP-IX: baseline, 28.3 +/- 0.85 sinusoids/mm; 10 minutes after SnPP-IX, 23.1 +/- 1.09 sinusoids/mm; P <.05). Intravital microscopy showed narrowing of failing sinusoids colocalizing with stellate cells after blockade of the HO pathway. Blockade of ET(A/B) receptors attenuated the increase in portal resistance (shock/bosentan/SnPP-IX, 0.29 +/- 0.051 mm Hg. min. mL(-1)) and prevented sinusoidal perfusion failure (shock/bosentan/SnPP-IX: baseline, 28.2 +/- 0.97 sinusoids/mm; 10 minutes after SnPP-IX, 28.8 +/- 1.18 sinusoids/mm) as well as sinusoidal narrowing. In conclusion, a functional interaction of the up-regulated vasodilatory HO system and the vasoconstrictor ET-1 on the sinusoidal level exists under stress conditions. Both mediator systems affect sinusoidal diameter via direct action on hepatic stellate cells in vivo.

Functional recovery of preserved livers following warm ischemia: improvement by machine perfusion preservation.

BACKGROUND: Hypothermic machine perfusion preservation has the potential to relieve the current donor shortage problem by reclaiming and preserving marginal donor organs including those from viable non-heart-beating donors. A number of problems exist with the current machine perfusion technology for preserving livers, and much research is needed to determine the clinical impact of this technology in preserving non-heart-beating donor livers. METHODS: This study was conducted to compare the poststorage function and microcirculation of simple cold stored and machine perfusion preserved livers that had experienced 30 min of warm ischemia followed by a 10 hr preservation period. In an isolated rat liver perfusion model, lactate dehydrogenase activity, indocyanine green secretion, and portal pressure values were determined at major time points. An intravital microscopy was conducted to assess microcirculation. RESULTS: The results showed an increase in flow homogeneity of machine perfused livers, which correlated with the reduction in portal pressure when compared with simple cold storage (5.4+/-0.4 vs. 8.7+/-0.6 mm Hg). A reduction in lactate dehydrogenase levels in the perfusate (333+/-22 vs.103+/-8 U/L) and an increase in bile production of the machine perfused livers (4.9+/-0.5 vs. 33.2+/-1.7 microg/min/g liver) and indocyanine green secretion (11.7+/-1.7 vs. 21.2+/-2.1 Abs/g bile) were observed at all time points (mean+/-SE of final point given). Intravital microscopic examination indicated that large regions of non flow, as indicated by the absence of fluorescein isothiocyanate-labeled albumin, were observed in the simple cold stored tissue, whereas machine perfused liver showed increase flow homogeneity. Values of bile production, indocyanine secretion, and cellular damages were comparable with controls. Histologic examination confirmed that simple cold stored tissue displayed increased vacuolization, and machine perfused tissue showed regions of normal hepatic structure. CONCLUSION: These results suggest that machine perfusion for 10 hr improves both poststorage function and microcirculation while reducing cellular damage of liver tissue that has experienced 30 min of warm ischemia, when compared with simple cold storage. Further studies need to be conducted, but this study suggests that machine perfusion preservation has the potential to reclaim and preserve liver tissues after warm ischemic insult.

Hepatic vascular response to elevated intraperitoneal pressure in the rat.

The rat is increasingly being used to study the physiological response to elevated intra-abdominal pressure (IAP) during laparoscopic surgery. Although decreased portal venous flow associated with the elevated IAP has been reported in large animals, little information is available in rats. Furthermore, the relative blood flow changes in the hepatic artery and portal vein have not been reported. Therefore, this study was performed to elucidate the change in systemic and splanchnic circulation, including hepatic arterial and portal venular flow, during pneumoperitoneum in rats. Sprague-Dawley rats were assigned into either a ventilated or nonventilated group and then subjected to various levels of IAP (0, 5, 10, and 20 mm Hg) using carbon dioxide gas. At each pressure, both cardiac output and splanchnic organ flow were determined using fluorescent microspheres. There was no obvious hemodynamic difference between the ventilated and nonventilated groups. Mean arterial pressure and cardiac index were significantly lower with 20 mm Hg of IAP compared to 0 mm Hg in both groups. Flow to the spleen, stomach, duodenum, total intestine, and portal vein was all decreased by increasing IAP (P < 0.05 at 20 mm Hg compared to 0 mm Hg) and was significantly correlated to the decrease in cardiac index. However, the hepatic arterial flow was relatively preserved throughout all levels of IAP, suggesting activation of the hepatic arterial buffer response. We conclude that the decreased splanchnic flow during pneumoperitoneum largely depends on the decreased cardiac index. Hepatic artery flow, however, is selectively preserved and may provide protection for liver function during sustained elevations in IAP.

Endothelin 1 impairs oxygen delivery in livers from LPS-primed animals.

Endothelin 1 (ET-1) is a potent vasoactive peptide that acts at sinusoidal and extrasinusoidal sites in the liver. Sensitivity to ET-1 increases in LPS-primed animals and is associated with impaired liver microcirculation in these animals. We hypothesized that LPS priming leads to an exacerbation in the impaired oxygen delivery in response to intraportal infusion of ET-1. Rats were studied 24 h after LPS injection (1 mg/kg, i.p.). Surface PO2 was determined using a recently developed technology of O2 mapping. The baseline portal pressure was higher in LPS-primed animals (P < 0.05), and increased to'similar magnitude as sham animals after a 10-min infusion of ET-1. The resultant portal pressure remained elevated in LPS compared to sham animals. There was no significant difference in baseline mean arterial pressure, and no significant systemic response to ET-1 in either group. In contrast to the macrohemodynamic, the decrease in tissue surface PO2 in response to ET-1 infusion was potentiated by LPS treatment (increased from baseline levels 33.8+/-9 to 46.8+/-8.3 in sham; 42.3+/-9.1 to 69+/-6.5 gray scale units in LPS; P < 0.01, sham vs. LPS) at end of infusion of ET-1 for 10 min. This indicates tissue hypoxia in response to ET-1, which is exacerbated in livers from LPS-primed animals compared to sham. Frequency distribution analysis showed a shift in mode from lower intensity (higher PO2) to areas with higher fluorescent intensity ranges (lower PO2), indicating areas with shut down in perfusion in LPS-treated animals. In the whole liver, ET-1 suppressed oxygen consumption, and this response was potentiated by LPS pretreatment. We propose that ET-1 impairs oxygen delivery in the liver during endotoxemia, resulting in areas of focal hypoxia. This response is possibly due to potentiated action of ET-1 at both sinusoidal and extrasinusoidal sites in the liver during endotoxemia.

LPS-induced imbalanced expression of hepatic vascular stress genes in cirrhosis: possible mechanism of increased susceptibility to endotoxemia.

Cirrhosis predisposes the liver to secondary stresses such as endotoxemia possibly via dysregulation of the hepatic portal circulation secondary to imbalanced upregulation of vascular stress genes. In this study we determined the effect of cirrhosis on hepatic vasoregulatory gene expression in response to endotoxin (LPS, i.p., 1 mg/kg). Cirrhosis was induced by bile duct ligation (BDL) for 21 days in male Sprague-Dawley rats. Plasma and liver samples were taken 6 h following an injection of LPS for alanine aminotransferase (ALT) assays and RT-PCR analysis of mRNA levels for genes of interest: endothelin (ET-1), its receptors ET(A) and ET(B), endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), and heme oxygenase-1 (HO-1). ALT release increased by 5.5-fold in the BDL animals and 9.9-fold in BDL + LPS compared to sham. ET-1 mRNA was increased by either LPS or BDL treatment alone and increased significantly more in BDL + LPS compared to sham + LPS. mRNA levels for ET(B) receptors showed no change, whereas ETA transcripts decreased in BDL animals compared to sham, with no significant difference between the saline and LPS treatment groups. The resultant increased ratio of ET(B) over ET(A) in BDL animals was reflected functionally in the portal pressure responses to ET(A) and ET(B) agonists ET-1 and IRL-1620 (a specific ETB receptor agonist). The pressor response to ET-1 was attenuated, while the response to IRL-1620 was similar in BDL and sham. eNOS mRNA levels did not increase in response to either BDL or LPS or a combination of both compared to sham. The increase in iNOS mRNA was attenuated in BDL + LPS compared to sham + LPS. HO-1 expression increased significantly in sham + LPS, but failed to increase in BDL + LPS. Taken collectively, significantly greater induction of the constrictor ET-1 over the dilation forces (i.e., eNOS, iNOS, and HO-1) was observed in BDL + LPS. This suggests a compromised ability of the cirrhotic liver to upregulate sufficient dilatory forces to counterbalance the constrictive effect of ET-1 upon a secondary insult of endotoxemia. These results may partly explain the increased susceptibility of cirrhotic livers to injury as a result of endotoxemia.

Endothelin receptor remodeling induces the portal venous hyper-response to endothelin-1 following endotoxin pretreatment.

The purpose of this study was to determine the changes in endothelin (ET) receptor subtype expression and their functional significance after endotoxin pretreatment. Rats were pretreated with lipopolysaccharide (LPS) or sterile saline (control). After 24 h, liver samples were homogenized and competitive receptor binding assays were performed. There was no significant difference in ET receptor binding affinity between the control and LPS groups. However, the receptor subtype density showed a significant increase in ET(B) receptors in LPS-treated rats, whereas the amount of ET(A) receptors was almost identical between the two groups. In control, almost all ET receptors (95%) were displaced by using combined ET(A) antagonist (BQ-610) and ET(B) agonist (IRL-1620) as competitors, whereas only 80% (P < 0.05 versus control) was displaced in LPS group, raising the possibility of novel type of ET receptor expression. An infusion of ET(B) agonist (Sarafotoxin 6c, S6c) through portal vein in isolated perfused livers produced the same pressure response in both LPS and control groups; however, the portal pressure increase in response to the ET-1, which binds all ET receptors, was significantly potentiated in LPS-treated rats compared to controls. We conclude that altered regulation of ET receptors, in particular, the appearance of ET binding capacity that is not displaced by ET(A) or ET(B) competitors, may explain the hyper-response of the portal venous system to ET-1 during endotoxemia.