Natalie Sonin

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.

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.

Altered endothelin receptor subtype expression in hepatic injury after ischemia/reperfusion.

This study was performed to determine whether ischemia/reperfusion (I/R) injury in rat liver results in alterations in endothelin receptor expression. Hepatic ischemia was produced in rats for 60 min followed by 6 or 24 h reperfusion. Portal inflow pressure was increased (7.38+/-0.60 mmHg) at 24 hours after reperfusion. Serum ALT increased significantly at both 6 and 24 h (6 h; 258.3+/-74.3, 24 h; 243.1+/-74.8 IU/L). Portal vascular response to an endothelin-B receptor agonist (IRL 1620) was significantly increased in the I/R livers compared to control and this was potentiated by L-NAME. IRL 1620 also caused LDH release from I/R livers but not controls. LDH release after IRL 1620 in I/R livers correlated with increased portal pressure response. To determine whether the altered response might be the result of altered endothelin receptor expression, livers were harvested after reperfusion and total endothelin binding sites were determined by competitive binding with ET-1. Proportion of endothelin receptor subtypes (ET(A)/ET(B)) was determined using the ET(A) antagonist BQ-610 (1 microM) and ET(B) agonist IRL-1620 (100 nM). There were no significant changes in Kd but Bmax for endothelin-1 was decreased in I/R group especially non-ischemic lobe at 24 h. ET(A) receptors were significantly decreased whereas ET(B) receptors were increased. These changes were more pronounced at 24 h after reperfusion than at 6 h. Interestingly, the changes in ET receptors was observed identically both in ischemic and non-ischemic lobes (ischemic lobe ET(A) 41.9%, ET(B) 51%; non-ischemic lobe ET(A) 38.8%, ET(B) 49.5%). These results indicate that the major functional endothelin receptor subtype upregulated in I/R is the ET(B) receptor and that this upregulation may contribute to microvascular dysregulation and hepatic injury.

Ischemia/reperfusion induces an increase in the hepatic portal vasoconstrictive response to endothelin-1

Microvascular impairment observed during reperfusion following ischemia (IR) is a major determinant of the development of liver injury. Previous studies have shown that hyper-responsiveness to endothelin-1 (ET-1) contributes to microvascular dysfunction following a primarily inflammatory stress induced by endotoxin. The present study investigates whether a similar hypercontractile response to ET-1 occurs in the hepatic portal system of IR rats. Pentobarbital-anesthetized Sprague-Dawley rats underwent liver ischemia of the left and medial lobes for 60 min (IR: n = 8) or a sham operation (n = 8). Six hours after reperfusion, the liver was isolated and perfused through the portal vein. Baseline portal pressure (Pp), portal flow (Qp), and sinusoidal diameter (Ds) were measured before and 3 and 10 min after adding ET-1 (1 nM). In baseline, IR livers had a significantly greater Pp, portal resistance, and Ds than sham. ET-1 significantly increased Pp and portal resistance and significantly decreased Qp and Ds in IR and sham rats. However, these effects were significantly greater in IR. The results of the present study demonstrate that IR increases the porto-hepatic contractile response to ET-1, which may further sensitize the portal circulation to elevated ET-1 and may be a prominent contributor to the development of microvascular impairment following IR.

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.

Inhibition of poly(ADP-ribose) synthetase improves vascular contractile responses following trauma-hemorrhage and resuscitation.

Hyporeactivity of vessels to constrictor agents is thought to contribute to cardiovascular decompensation following trauma-hemorrhage and resuscitation. In this study, we determined if inhibition of poly(ADP-ribose) synthetase (PARS) activity prevented the development of vascular hyporeactivity in rats following trauma-hemorrhage and resuscitation. Trauma consisted of a laparotomy that was closed and rats were hemorrhaged into a reservoir containing citrate to 40 mm Hg for 90 min. Resuscitation included 2/3 of the shed blood plus 2 1/3 of the shed volume as Ringers lactate. Sham animals received the laparotomy and were time-matched. Induction of iNOS was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Aortic rings isolated 6 h after the initiation of hemorrhage (4.5 h after resuscitation) showed decreased responsiveness to norepinephrine (peak developed tension 0.31+/-0.01 g/mg tissue) compared with sham rings (0.43+/-0.02 g/mg tissue), but no change in EC50 for this response (approximately 5x10(-8) M). Addition of the PARS inhibitor, 3-aminobenzamide, at the onset of resuscitation prevented the decrease in response of aortic rings. The addition of the structural analogue, 3-aminobenzoic acid, which does not inhibit PARS, did not prevent the decrease in vascular reactivity. These agents did not alter vascular responses to norepinephrine in sham animals. iNOS induction was not associated with depressed contractile function. These results indicate that decreased vascular reactivity was prevented by inhibition of PARS and that PARS activation was independent of iNOS induction following trauma-hemorrhage and resuscitation.

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 ETB receptors in LPS-treated rats, whereas the amount of ETA receptors was almost identical between the two groups. In control, almost all ET receptors (95%) were displaced by using combined ETA antagonist (BQ-610) and ETB 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 ETB 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 ETA or ETB competitors, may explain the hyper-response of the portal venous system to ET-1 during endotoxemia.

Altered endothelin receptor expression in prehepatic portal hypertension predisposes the liver to microcirculatory dysfunction in rats

BACKGROUND/AIMS Endothelin (ET) is one of the most active vascular regulators in the liver. It is unknown how partial portal vein ligation (PPVL) induced prehepatic portal hypertension influences the response of the liver to ET and its agonists. Therefore, this study was conducted to determine the expression of ET receptors and its functional significance after PPVL. METHODS Competitive receptor binding study and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) were performed using liver homogenates after 2 weeks of PPVL or sham operation in rats. Hepatic microcirculation was evaluated in vivo using intravital microscopy. RESULTS Although there was no significant difference in dissociation constant (Kd) and total amount of receptors (Bmax) between sham and PPVL, the proportion of ET(B) receptor was significantly increased in PPVL. RT-PCR analysis confirmed the up-regulation of ET(B) receptors demonstrated by the competitive receptor binding assay. In the functional study, infusion of ET(B) agonist (IRL 1620) in a low dosage did not change the hepatic microcirculation in sham but strongly constricted the sinusoids leading to a reduction of sinusoidal perfusion in PPVL. CONCLUSIONS These results suggest that prehepatic portal hypertension may predispose the hepatic microcirculation to dysregulation in stress conditions where ET is upregulated.