Steve Keller

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 (VRBC) 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 ETA and ETB, inducible and constitutive nitric oxide (NO) synthase (iNOS and eNOS, respectively), and heme oxygenase 1 (HO-1). After CLP, both ET-1 and ETB mRNA increased, whereas ETA 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.

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 ETA and ETB, 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 ETB 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 ETB over ETA in BDL animals was reflected functionally in the portal pressure responses to ETA and ETB 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.

Perflubron emulsion improves hepatic microvascular integrity and mitochondrial redox state after hemorrhagic shock.

Hemorrhagic shock is associated with decreased systemic oxygen delivery, but also with impaired microvascular perfusion, which can result in diminished local oxygen availability even in the presence of adequate cardiac output after resuscitation. Beside surgical interventions to control blood loss, transfusion of stored packed red blood cells represents the current standard of care in the management of severe hemorrhagic shock. Because stored red blood cells are less deformable and show a higher O2 affinity that affects the O2 off-load to tissues, perfluorocarbon-based artificial oxygen carriers might improve local O2 delivery under these conditions. To test this, rats were subjected to hemorrhagic shock (1 h, mean arterial pressure [MAP] 30-35 mmHg) and were resuscitated with fresh whole blood, pentastarch, stored red blood cells, perflubron emulsion (2.7 and 5.4 g/kg body weight) together with pentastarch, or stored red blood cells together with 2.7 g/kg perflubron emulsion. Hepatic microcirculation, tissue oxygenation, and mitochondrial redox state were investigated by intravital microscopy. In addition, hepatocellular function and liver enzyme release were determined. After hemorrhagic shock and resuscitation with perflubron emulsion, volumetric sinusoidal blood flow was significantly increased compared with resuscitation with stored red blood cells. Furthermore, resuscitation with perflubron emulsion resulted in higher hepatic tissue PO2 and normalized mitochondrial redox potential, which was accompanied by lessened hepatocellular injury as well as improved liver function. These results indicate that, in this model of hemorrhagic shock, asanguineous fluid resuscitation with addition of perflubron emulsion is superior to stored blood or pentastarch alone with respect to increased local O2 availability on the cellular level. This effect is primarily due to improved restoration of hepatic microcirculatory integrity.

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 μm/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 μm 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.

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

ABSTRACT 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 (30mg/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.

Induction of biphasic changes in perfusion heterogeneity of rat liver after sequential stress in vivo.

Trauma and subsequent sepsis lead to hepatic microcirculation disruption through various molecular mechanisms in which endothelin-1 (ET-1) plays a pivotal role. These stresses are thought to alter hepatic perfusion, heterogeneously leading to a mismatch of oxygen supply and demand. We hypothesize that mild remote stresses prime the liver to sequential sepsis through direct effects on the hepatic lobular flow distribution. We also propose to investigate the extent and the localization of the stress-induced microcirculation disruption. Sprague-Dawley rats were randomly divided into four experimental groups: sham, femur fracture (FFX), cecal ligation and puncture (CLP), and sequential stress (SS). Hepatic intravital microscopy was performed for in vivo assessment of the liver microcirculation flow distribution under baseline and after ET-1 infusion. Red blood cell motion distribution was used to quantify intralobular and interlobular heterogeneity of perfusion (HoP). Intralobular HoP, which reflects lobular regulation sites, was significantly increased in the FFX and CLP groups, but was not changed or decreased in the SS group compared with control. ET-1 infusion exerted opposite effects depending on the pathological condition, further increasing the difference between groups. SS induced decrease in intralobular HoP, contrasted with a significant increase in interlobular HoP, suggesting multiple disruption sites. Our data suggest that increased intralobular HoP may be indicative of a compensatory response to moderate stress; its decrease under sequential stress conditions corresponds with a total breakdown of hepatic lobular flow regulation. This may be another instance of the rich variability characteristic of normal physiology that “decomplexifies” under critical decompensated conditions.

Activated protein C restores hepatic microcirculation during sepsis by modulating vasoregulator expression.

Activated protein C (aPC) promotes fibrinolysis while inhibiting coagulation and inflammation. In septic patients, aPC levels are depleted, and aPC treatment has emerged as a therapeutic option. To better understand the mechanism(s) by which aPC improves survival in sepsis, we sought to determine the effect of aPC treatment on hepatic vasoactive gene and protein expression, leading to changes in hepatic vascular responsiveness in a septic animal model. Under anesthesia, rats underwent sham or cecal ligation and puncture followed by aPC treatment (1 mg/kg, twice daily, i.v.). Treatment with aPC significantly decreased hepatic endothelin 1 (ET-1)/ET A receptor mRNA and protein expression. To determine the effect of aPC on hepatic microvasculature, ET-1-induced changes in liver microcirculation were assessed by intravital microscopy. This approach demonstrated aPC significantly improved hepatic perfusion index in the animals that underwent cecal ligation and puncture in the absence of significant changes in portal venous pressure. Furthermore, although aPC did not affect ET-1-dependent sinusoidal vasoconstriction, aPC induced hepatoprotective effects via enhanced red blood cell velocity. Collectively, these data demonstrate aPC ameliorates ET-1-dependent changes in hepatic microcirculation and improves hepatic function in the setting of sepsis.