Walid S. Kamoun

LPS inhibits endothelin-1-induced endothelial NOS activation in hepatic sinusoidal cells through a negative feedback involving caveolin-1.

During endotoxemia, liver microcirculation disruption is characterized by a hypersensitivity to the constrictor effects of endothelin 1 (ET‐1). The shift of ET‐1–mediated effects toward vasoconstriction may result from depressed ET‐1–mediated vasodilation through decreased ET‐1–induced nitric oxide (NO) production. We have previously shown that lipopolysaccharide (LPS) pretreatment abrogates ET‐1–induced endothelial nitric oxide synthase (eNOS) translocation, but its effects on eNOS activation are yet to be determined. Our aim was to assess the effects of LPS on ET‐1–mediated eNOS activation in hepatic sinusoidal endothelial cells (SECs) and to investigate the molecular mechanisms involved. SECs were treated with LPS (100 ng/mL) for 6 hours followed by 30 minutes ET‐1 (10 nmol/L) stimulation. LPS significantly inhibited ET‐1–mediated eNOS activation. This inhibition was associated with upregulation of Caveolin‐1 (CAV‐1) and a shift in ET‐1–mediated eNOS phosphorylation from an activation (Ser1177) to an inhibition (Thr495). LPS treatment has been shown to induce ET‐1 expression and secretion from endothelial cells. We therefore investigated the role of endogenous ET‐1 in the inhibition of ET‐1 activation of eNOS after LPS. Antagonizing ET‐1 effects and blocking its activation in LPS pretreated SECs decreased the LPS‐induced overexpression of CAV‐1 as well as the inhibition of ET‐1–induced NOS activity. Furthermore, 6 hours of ET‐1 treatment exerted the same effects on eNOS activity, phosphorylation, and CAV‐1 expression as LPS treatment. In conclusion, LPS‐induced suppression of ET‐1–mediated eNOS activation is ET‐1 dependent and suggest a pivotal role of CAV‐1 in eNOS induction inhibition under stress. (HEPATOLOGY 2006;43:182–190.)

Chronic ethanol sensitizes the liver to endotoxin via effects on endothelial nitric oxide synthase regulation

In vivo studies have shown that chronic alcohol consumption sensitizes the liver to endotoxemic shock, leading to liver microcirculation disruption. In the present study, we investigated the molecular mechanisms involved, focusing on endothelial nitric oxide synthase (eNOS) activity and regulation, which represents one of the major vasodilatory pathways. Male Sprague-Dawley rats were fed an alcohol liquid diet or a control isocaloric diet for 5 weeks. Priming effects of ethanol were studied in a model with or without a 24-h LPS treatment (1 mg/kg body weight). At the end of the diet, liver tissue was harvested for western blot, reverse transcriptase-PCR, histological analysis, and immunostaining and blood for serum alanine aminotransferase analysis. Chronic ethanol and LPS alone induced a mild hepatitis and infiltration, respectively. Combined, LPS and chronic ethanol feeding showed a synergistic effect on the liver, leading to extensive steatohepatitis with extensive focal necrosis associated with significantly higher levels of serum ALT. Chronic ethanol and LPS significantly inhibited eNOS activity, but exerted their effects through different mechanisms. Caveolin-1, an eNOS inhibitory protein, was upregulated after LPS and chronic alcohol consumption. Additionally, chronic alcohol consumption down-regulated endothelin B receptor, eNOS protein levels, and eNOS phosphorylation. In conclusion, chronic ethanol consumption and LPS share a similar pathophysiology and both lead to the impairment of eNOS activity, but through distinct molecular mechanisms. The presence of focal necrosis in a mild stress model could provide a good animal study to investigate the advanced stages of alcoholic liver diseases.

Differential Effects of Oxidative Stress on Hepatic Endothelial and Kupffer Cell Eicosanoid Release in Response to Endothelin‐1

Objective: The vasoconstrictor endothelin‐1 can induce vasomodulators release like nitric oxide in the liver. Here the authors explored whether endothelin‐1 can stimulate endothelial and Kupffer cells release of other vasomodulators under normal and stress conditions.

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.

LPS Inhibits Endothelin-1-Mediated eNOS Translocation to the Cell Membrane in Sinusoidal Endothelial Cells

Objective: The objectives of this study were to develop a model for studying endothelin‐1‐mediated eNOS regulation in cultured sinusoidal endothelial cells and determine the effect of endothelin‐1 and endotoxin (LPS) on eNOS localization.

Liver Microcirculation Analysis by Red Blood Cell Motion Modeling in Intravital Microscopy Images

Intravital microscopy has been used to visualize the microcirculation by imaging fluorescent labeled red blood cells (RBCs). Traditionally, microcirculation has been modeled by computing the mean velocity of a few, randomly selected, manually tracked RBCs. However, this protocol is tedious, time consuming, and subjective with technician related bias. We present a new method for analyzing the microcirculation by modeling the RBC motion through automatic tracking. The tracking of RBCs is challenging as in each image, as many as 200 cells move through a complex network of vessels at a wide range of speeds while deforming in shape. To reliably detect RBCs traveling at a wide range of speeds, a window of temporal template matching is applied. Then, cells appearing in successive frames are corresponded based on the motion behavior constraints in terms of the direction, magnitude, and path. The performance evaluation against a ground truth indicates the detection accuracy up to 84% TP at 6% FP and a correspondence accuracy of 89%. We include an in-depth discussion on comparison of the microcirculation based on motion modeling from the proposed automated method against a mean velocity from manual analysis protocol in terms of precision, objectivity, and sensitivity.