Mark G. Clemens

Protective role of endogenous carbon monoxide in hepatic microcirculatory dysfunction after hemorrhagic shock in rats.

Maintenance of hepatic microcirculatory flow after ischemia of the liver is essential to prevent hepatic dysfunction. Thus, we determined the differential role of carbon monoxide (CO) and nitric oxide (NO) in the intrinsic control of sinusoidal perfusion, mitochondrial redox state, and bile production in the isolated perfused rat liver after hemorrhagic shock. Administration of tin protoporphyrin-IX (50 microM), a specific inhibitor of the CO generating enzyme heme oxygenase, caused a decrease in sinusoidal flow that was more pronounced after shock compared with sham shock, as determined by in situ epifluorescence microscopy. This was associated with a shift in hepatocellular redox potential to a more reduced state (increased fluorescence intensity of reduced pyridine nucleotides in hepatocytes, decreased acetoacetate/beta-hydroxybutyrate ratio in the perfusate) and a profound reduction in bile flow. In sharp contrast, the preferential inhibitor of the inducible isoform of NO synthase S-methylisothiourea sulfate (100 microM) did not affect sinusoidal flow, hepatic redox state, or function. This indicates that 1.) endogenously generated CO preserves sinusoidal perfusion after hemorrhagic shock, 2.) protection of the hepatic microcirculation by CO may serve to limit shock-induced liver dysfunction, and 3.) in contrast to CO, inducible NO synthase-derived NO is of only minor importance for the intrinsic control of hepatic perfusion and function under these conditions.

Microcirculatory failure determines lethal hepatocyte injury in ischemic/reperfused rat livers.

The contribution of microcirclatory failure to ischemia/reperfusion injury in isolated perfused rat livers was investigated using intravital epifluorescence videomicroscopy. The degree of microvascular shut-down during reperfusion was modulated by the reperfusion conditions: flow-controlled (10 ml/min), in which microcirculatory failure is minimized by maintenance of constant flow through the liver, and pressure-controlled, in which microvascular shut-down is allowed to occur. Livers underwent 60 min of ischemia, 90 min of ischemia, or no ischemia (control). Perfused sinusoids and dead hepatocytes were quantified in 10 standardized microscopic fields (9000 μ2) per liver during off-line video playback. With flow-controlled reperfusion, microvascular (sinusoid) shut-down was largely avoided; a maximum of 21% of the sinusoids failed to conduct flow. Pressure-controlled reperfusion, however, resulted in early and severe shut-down. A sinificant decrease of approximately 20–30% was found after 60 min of ischemia and 30 min of reperfusion, while, after 90-min ischemia and 90-min reperfusion, 90% of the sinusoids failed to conduct flow. The appearance of dead hepatocytes correlated well with the number of perfused sinusoids (r = −0.78 for flow controlled, r = −0.97 for pressure-controlled). Only an occasional dead hepatocyte was observed with control perfusion, while up to 50% stained with propidium iodide following 90-min ischemia and 90-min reperfusion under pressure-controlled conditions. These results indicate that loss of sinusoidal flow can be ameliorated by flow-controlled reperfusion; moreover, hepatocyte necrosis during reperfusion is highly dependent upon the integrity of the microcirculation.

Patterns of vasoregulatory gene expression in the liver response to ischemia/reperfusion and endotoxemia.

Oxidative stress and inflammatory reactions associated with stresses that may lead to shock promote hepatic microcirculatory dysfunction, which may lead to hepatic injury. Because altered liver microcirculation may result from an imbalance in the expression of stress-induced vasoactive mediators, our study was conducted to investigate changes in the expression of genes encoding endothelin-1 (ET-1), its receptors, ET(A) and ET(B), heme-oxygenase 1 (HO-1), and inducible nitric oxide synthase (iNOS), using two different rat models of liver stress: ischemia/reperfusion of the liver and lipopolysaccharide (LPS)-induced endotoxemia. In ischemia/reperfusion experiments, rats were subjected to 1 h hepatic ischemia, followed by 6 h of reperfusion. Endotoxemia was induced by i.p. injection of LPS (1 mg/mL/kg body weight); rats were studied after 6 h. mRNA levels were estimated using semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on total RNA samples prepared from experimental and sham control rat livers. In the ischemic reperfused livers the levels of mRNA for ET-1, ET(B), HO-1, and iNOS were significantly elevated. The fold increase versus sham was 2.5+/-1.1 (ET-1), 2.1+/-1.3 (ET(B)), 2.1+/-.8 (HO-1), and 6.4+/-3.9 (iNOS). In contrast, the expression of ET(A) receptor gene was reduced after ischemia/reperfusion (to 73+/-1% of sham). In the separate experiments we analyzed the same mRNAs levels after 1 h of ischemia (no reperfusion), and did not detect any changes. During endotoxemia we observed a marked increase in iNOS mRNA level (>24-fold), as well as a marked elevation of the other four mRNAs. The fold increase versus sham was 6.1+/-1.7, ET-1); 1.5+/-.3 (ET(A)); 1.6+/-.4 (ET(B)); and 2.4+/-.34 (HO-1). These results show that liver stress, induced by ischemia/reperfusion or LPS injection have characteristic patterns of vasoregulatory genes expression indicating that, although both stresses result in an increase in specific vascular reactivity, different pathways are involved in inducing the hepatic vascular stress response.

Survival transplantation of preserved non???heart-beating donor rat livers: preservation by hypothermic machine perfusion1

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.

REMODELING OF HEPATIC MICROVASCULAR RESPONSIVENESS AFTER ISCHEMIA/REPERFUSION

Although there is substantial evidence suggesting that the integrity of the microcirculation is an important determinant of tissue viability during reperfusion after ischemia in the liver, as well as other tissues, the mechanisms responsible for microvascular failure are not fully understood. It is now recognized that the microvascular response to reperfusion, similar to the whole organism response to shock, can consist of either a rapid exacerbation of injury after a severe ischemic episode or, alternatively, a more slowly developing alteration in responsiveness that occurs after a less severe insult. In the more slowly developing response, the alterations in vascular status are the result of up-regulation of stress-induced vascular mediators such as endothelin, nitric oxide synthase (NOS), and heme oxygenase, as well as changes in the reactivity of the effector cells to the mediators. The mechanisms for change in reactivity of vascular cells range from changes in receptor expression to overt phenotypic transformation, as can occur in the hepatic stellate cells in response to repeated injury. When maintained in balance, these counteracting constrictor and dilator influences can be protective; however, local imbalance can result in focal ischemia, thus propagating the injury. Thus, the remodeling of the hepatic microvascular responsiveness during reperfusion after ischemia may serve as a useful paradigm for consideration of the overall response of the organism to shock.

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 ETA/B antagonist bosentan (10 mg/kg intravenously). Subsequently, the HO pathway was blocked in all animals by tin‐protoporphyrin (SnPP)‐IX (50 μmol/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 < 0.05) 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 < 0.05). Intravital microscopy showed narrowing of failing sinusoids colocalizing with stellate cells after blockade of the HO pathway. Blockade of ETA/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. (HEPATOLOGY2002;36:1453–1465).

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.

Posttranscriptional regulation of connexin 32 expression in liver during acute inflammation

Gap junctions mediate the communication between adjacent cells in tissues. In the liver, connexin 32 (Cx32) subunits make up the predominating gap junctions. The expression of Cx32 gene has been observed to be down‐regulated in response to inflammatory states and during liver regeneration. In the present study we attempt to elucidate the molecular mechanisms underlying the down‐regulation of the Cx32 expression during acute inflammation. A decrease in the level of Cx32 mRNA in rat liver occurred between 3 and 6 h after intravenous administration of bacterial lipopolysaccharide (LPS), simultaneously with the induction of an acute inflammatory response characterized by an increase in the level for β‐fibrinogen and a reduction of phosphoenolpyruvate carboxykinase mRNA. The reduction in Cx32 steady‐state mRNA levels appears to occur at the posttranscriptional level, since the rate of degradation of this message seems to be higher than the rate of transcription of the gene. Degradation of Cx32 mRNA was blocked by the administration of actinomycin D, but not by cycloheximide, prior to injection of LPS. The stabilization of Cx32 message by actinomycin D correlated with the preservation of Cx32 on the cell surface, which otherwise disappears after administration of LPS alone. These results suggest that cellular communication via gap junctions could be regulated at the level of gene expression, by a posttranscriptional mechanism, during acute inflammatory states.

Hepatic intercellular communication in shock and inflammation.

The liver is well recognized as a target for injury during low flow or inflammatory states. Functionally, the result is both metabolic and host defense dysfunction. Although the liver is clearly responsive to changes in systemic levels of various mediators, it is becoming apparent that substantial changes occur within the liver that are not directly dependent on extrahepatic factors. This is the result of complex interactions among the various cell types that exist in a highly organized arrangement within the functional subunit of the liver. The purpose of this review is to summarize the structural relationships which form the basis for this system of cell-cell communication and their functional implications both in the normal liver and during both low-flow and normal-flow inflammatory states.

Chronic ethanol consumption exacerbates liver injury following hemorrhagic shock: role of sinusoidal perfusion failure.

Although the deleterious effect of chronic ethanol consumption on subsequent stressful events has long been recognized, the pathophysiological mechanisms are incompletely understood. This study tested whether chronic ethanol consumption in doses that increase sinusoidal contractility increases susceptibility to hepatic microvascular failure and liver injury after hemorrhagic shock. Liver microcirculation was assessed by in vivo microscopy during hemorrhage and up to 24 h after onset of resuscitation and was compared with liver histology and serum enzyme levels. Mean sinusoidal blood flow was neither impaired by chronic ethanol feeding at baseline nor during hemorrhage and early resuscitation. However, failure of individual sinusoids to conduct flow was observed more frequently after fluid resuscitation in ethanol-fed animals (e.g. at 1 h after onset of volume therapy: 26% of sinusoids) than in controls (11%), reflecting substantial flow heterogeneity. Failing sinusoids had substantially smaller diameters than sinusoids conducting flow with a more profound and sustained response in ethanol-fed rats. At 24 h marked pericentral necrosis and increase in serum alanine aminotransferase levels were observed in six of nine surviving ethanol-fed animals but only in 1 of 10 pair fed controls and correlated with microvascular failure. These data suggest that early as well as late microvascular failure in this model of hemorrhagic shock and resuscitation is primarily mediated at the level of individual sinusoids. Chronic ethanol feeding exacerbates microvascular and hepatocellular injury after shock/resuscitation, probably involving increased sinusoidal contractile responsiveness.