Liver Sinusoidal Endothelial Cells.

The liver sinusoidal endothelial cell (LSEC) forms the fenestrated wall of the hepatic sinusoid and functions as a control post regulating and surveying the trafficking of molecules and cells between the liver parenchyma and the blood. The cell acts as a scavenger cell responsible for removal of potential dangerous macromolecules from blood, and is increasingly acknowledged as an important player in liver immunity. This review provides an update of the major functions of the LSEC, including its role in plasma ultrafiltration and regulation of the hepatic microcirculation, scavenger functions, immune functions, and role in liver aging, as well as issues that are either undercommunicated or confusingly dealt with in the literature. These include metabolic functions, including energy metabolic interplay between the LSEC and the hepatocyte, and adequate ways of identifying and distinguishing the cells.

Acute atorvastatin is hepatoprotective against ischaemia-reperfusion injury in mice by modulating eNOS and microparticle formation.

BACKGROUND & AIMS: Steatosis accentuates the severity of hepatic ischaemia-reperfusion injury (IRI); 'statins' (HMG-CoA reductase inhibitors) protect the heart and brain against post-ischaemic injury. We tested whether short-term administration of atorvastatin protects fatty livers in obese mice against IRI. METHODS: Mice with dietary or genetic simple steatosis (SS) or non-alcoholic steatohepatitis (NASH) were subjected to 60 min partial hepatic ischaemia/24 h reperfusion. Atorvastatin was injected intravenously (5 mg/kg) 1 h before IRI. Liver injury, Toll-like receptor-4 (TLR4), cytokines/chemokines, iNOS/eNOS expression, eNOS activity and thromboxane B2 (TXB2) production were determined. RESULTS: Ischaemia-reperfusion injury was exaggerated by two- to five-fold in SS and NASH compared with lean liver. Atorvastatin pretreatment conferred 70-90% hepatic protection in all animals. Atorvastatin increased post-ischaemic eNOS mRNA/protein and strikingly enhanced eNOS activity (by phospho-eNOS). It also attenuated microparticle (MP) production, NF-κB activation, significantly dampened post-ischaemic thromboxane B2 production, induction of TNF-α, IL-6, MIP-1a, MCP-1, GM-CSF and vascular cell adhesion molecule-1 (VCAM), with a resultant reduction on macrophage and polymorphonuclear neutrophil recruitment. Up-regulation of HMGB1 and TLR4 after IRI was marked in fatty livers; 1 h pretreatment with atorvastatin reduced HMGB1 and TLR4 expression in all livers. CONCLUSIONS: Acute (1 h) atorvastatin administration is highly hepatoprotective against IRI in NASH, fatty and lean livers. Key mechanisms include suppression of inflammation by prevention of NF-κB activation, microvascular protection via eNOS activation and suppression of TXB2 and MP release. Short-term intravenous statin treatment is a readily available and effective preventive agent against hepatic IRI, irrespective of obesity and fatty liver disease, and merits clinical trials in at-risk patients.

Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent.

BACKGROUND & AIMS: Free cholesterol (FC) accumulates in non-alcoholic steatohepatitis (NASH) but not in simple steatosis. We sought to establish how FC causes hepatocyte injury. METHODS: In NASH-affected livers from diabetic mice, subcellular FC distribution (filipin fluorescence) was established by subcellular marker co-localization. We loaded murine hepatocytes with FC by incubation with low-density lipoprotein (LDL) and studied the effects of FC on JNK1 activation, mitochondrial injury and cell death and on the amplifying roles of the high-mobility-group-box 1 (HMGB1) protein and the Toll-like receptor 4 (TLR4). RESULTS: In NASH, FC localized to hepatocyte plasma membrane, mitochondria and ER. This was reproduced in FC-loaded hepatocytes. At 40 µM LDL, hepatocyte FC increased to cause LDH leakage, apoptosis and necrosis associated with JNK1 activation (c-Jun phosphorylation), mitochondrial membrane pore transition, cytochrome c release, oxidative stress (GSSG:GSH ratio) and ATP depletion. Mitochondrial swelling and crystae disarray were evident by electron microscopy. Jnk1(-/-) and Tlr4(-/-) hepatocytes were refractory to FC lipotoxicity; JNK inhibitors (1-2 µM CC-401, CC-930) blocked apoptosis and necrosis. Cyclosporine A and caspase-3 inhibitors protected FC-loaded hepatocytes, confirming mitochondrial cell death pathways; in contrast, 4-phenylbutyric acid, which improves ER folding capacity did not protect FC-loaded hepatocytes. HMGB1 was released into the culture medium of FC-loaded wild type (WT) but not Jnk1(-/-) or Tlr4(-/-) hepatocytes, while anti-HMGB1 anti-serum prevented JNK activation and FC lipotoxicity in WT hepatocytes. CONCLUSIONS: These novel findings show that mitochondrial FC deposition causes hepatocyte apoptosis and necrosis by activating JNK1; inhibition of which could be a novel therapeutic approach in NASH. Further, there is a tight link between JNK1-dependent HMGB1 secretion from lipotoxic hepatocytes and a paracrine cytolytic effect on neighbouring cholesterol-loaded hepatocytes operating via TLR4.

Hepatic disposal of advanced glycation end products during maturation and aging.

UNLABELLED: Aging is characterized by progressive loss of metabolic and biochemical functions and accumulation of metabolic by-products, including advanced glycation end products (AGEs), which are observed in several pathological conditions. A number of waste macromolecules, including AGEs are taken up from the circulation by endocytosis mainly into liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs). However, AGEs still accumulate in different tissues with aging, despite the presence of this clearance mechanism. The aim of the present study was to determine whether the efficiency of LSECs and KCs for disposal of AGEs changes through aging. RESULTS: After intravenous administration of (14)C-AGE-albumin in pre-pubertal, young adult, middle aged and old mice, more than 90% of total recovered (14)C-AGE was liver associated, irrespective of age. LSECs and KCs represented the main site of uptake. A fraction of the (14)C-AGE degradation products ((14)C-AGE-DPs) was stored for months in the lysosomes of these cells after uptake. The overall rate of elimination of (14)C-AGE-DPs from the liver was markedly faster in pre-pubertal than in all post-pubertal age groups. The ability to eliminate (14)C-AGE-DPs decreased to similar extents after puberty in LSECs and KCs. A rapid early removal phase was characteristic for all age groups except the old group, where this phase was absent. CONCLUSIONS: Removal of AGE-DPs from the liver scavenger cells is a very slow process that changes with age. The ability of these cells to dispose of AGEs declines after puberty. Decreased AGE removal efficiency early in life may lead to AGE accumulation.

Effects of oxidized low-density lipoproteins on the hepatic microvasculature.

Oxidized low-density lipoproteins (oxLDLs) are involved in proinflammatory and cytotoxic events in different microcirculatory systems. The liver is an important scavenger organ for circulating oxLDLs. However, the interaction of oxLDL with the hepatic microcirculation has been poorly investigated. The present study was conducted to examine the effects of differently modified oxLDLs on the hepatic microvasculature. C57Bl/6J mice were injected intravenously with low-density lipoprotein (LDL), or LDL oxidized for 3 h (oxLDL(3)) or 24 h (oxLDL(24)), at doses resembling oxLDL plasma levels in cardiovascular disease patients. Radioiodinated ligands were used to measure blood decay and organ distribution, and nonlabeled ligands to evaluate microcirculatory responses, examined by in vivo microscopy 30-60 min after ligand injection, immunohistochemistry, and scanning and transmission electron microscopy. Mildly oxLDL (oxLDL(3)) was cleared from blood at a markedly slower rate than heavily oxLDL (oxLDL(24)), but significantly faster than LDL (P < 0.01). Injected oxLDLs distributed to liver. OxLDL effects were most pronounced in central areas of the liver lobules where oxLDL(3) elicited a significant (P < 0.05) reduction in perfused sinusoids, and both oxLDL(3) and oxLDL(24) significantly increased the numbers of swollen endothelial cells and adherent leukocytes compared with LDL (P < 0.05). OxLDL-treated livers also exhibited increased intercellular adhesion molecule (ICAM)-1 centrilobular staining. Electron microscopy showed a 30% increased thickness of the liver sinusoidal endothelium in the oxLDL(3) group (P < 0.05) and a reduced sinusoidal fenestration in centrilobular areas with increased oxidation of LDL (P for linear trend <0.05). In conclusion, OxLDL induced several acute changes in the liver microvasculature, which may lead to sinusoidal endothelial dysfunction.

The effects of old age on hepatic stellate cells.

Aging is associated with marked changes in the hepatic sinusoid, yet the effect of old age on hepatic stellate cells (HSC) has not been well described. Transmission electron microscopy and immunohistochemistry were used to study the effects of aging on HSC in livers from rats (3-4 mths versus 24-27 mths) and mice (2-3 mths versus 20-22 mths). Desmin-positive HSC doubled in old age in both mice and rats. Alpha-smooth muscle actin- (αSMA-) positive cells did not increase significantly and remained only a small percentage of desmin-positive cells. Electron microscopy revealed that old age is associated with HSC that have a substantial increase in the number of lipid droplets which are larger in diameter. There was also a marked increase of HSC that protruded into the sinusoidal lumen in old mice. In conclusion, old age is associated with hyperplasia of HSC that are not activated and are engorged with lipid droplets.

Liver sinusoidal endothelial fenestrations in caveolin-1 knockout mice.

OBJECTIVE: Fenestrations are pores in the liver sinusoidal endothelium that facilitate the transfer of particulate substrates between the sinusoidal lumen and hepatocytes. Fenestrations express caveolin-1 and have structural similarities to caveolae, therefore might be a form of caveolae and caveolin-1 may be integral to fenestration structure and function. Therefore, fenestrations were studied in the livers of caveolin-1 knockout mice. METHODS: Scanning, transmission and immunogold electron microscopic techniques were used to study the liver sinusoidal endothelium and other tissues in caveolin-1 knockout and wild-type mice. RESULTS: Comparison of fenestrations in wild-type and knockout mice did not reveal any differences on either scanning or transmission electron microscopy. The diameter of the fenestrations was not significantly different (74 +/- 13 nm knockout mice vs 78 +/- 12 nm wild-type mice) nor was the fenestration porosity (6.5 +/- 2.1 knockout vs 7.3 +/- 2.4% wild-type mice). In contrast, adipocytes and blood vessels in other tissues lacked caveolae in the knockout mice. Caveolin-1 immunogold of livers of wild-type mice indicated sparse expression in sinusoidal endothelial cells. CONCLUSIONS: The normal structure of fenestrations in the liver sinusoidal endothelium is not dependent upon caveolin-1 and fenestrations are not a form of caveolae.

Short-term therapy with peroxisome proliferation-activator receptor-alpha agonist Wy-14,643 protects murine fatty liver against ischemia-reperfusion injury.

UNLABELLED: Steatosis increases operative morbidity/mortality from ischemia-reperfusion injury (IRI); few pharmacological approaches have been protective. Using novel genetic/dietary models of nonalcoholic steatohepatitis (NASH) and simple steatosis (SS) in Alms1 mutant (foz/foz) mice, we characterized severity of IRI in NASH versus SS and lean liver and tested our hypothesis that the lipid-lowering effects of the peroxisome proliferation-activator receptor (PPAR)-alpha agonist Wy-14,643 would be hepatoprotective. Mice were subjected to 60-minute partial hepatic IRI. Microvascular changes were assessed at 15-minute reperfusion by in vivo microscopy, injury at 24 hours by serum alanine aminotransferase (ALT), and hepatic necrosis area. Injury and inflammation mediators were determined by way of immunoblotting for intercellular cellular adhesion molecule, vascular cellular adhesion molecule, p38, c-jun N-terminal kinase, IkappaB-alpha, interleukin (IL)-1a, IL-12, tumor necrosis factor-alpha (TNF-alpha) and IL-6, cell cycle by cyclin D1 and proliferating cell nuclear antigen immunohistochemistry. In foz/foz mice fed a high-fat diet (HFD) to cause NASH or chow (SS), IRI was exacerbated compared with HFD-fed or chow-fed wild-type littermates by ALT release; corresponding necrotic areas were 60 +/- 22% NASH, 29 +/- 9% SS versus 7 +/- 1% lean. Microvasculature of NASH or SS livers was narrowed by enormous lipid-filled hepatocytes, significantly reducing numbers of perfused sinusoids, all exacerbated by IRI. Wy-14,643 reduced steatosis in NASH and SS livers, whereas PPAR-alpha stimulation conferred substantial hepatoprotection against IRI by ALT release, with reductions in vascular cellular adhesion molecule-1, IL-1a, TNF-alpha, IL-12, activated nuclear factor-kappaB (NF-kappaB), p38, IL-6 production and cell cycle entry. CONCLUSION: NASH and SS livers are both more susceptible to IRI. Mechanisms include possible distortion of the microvasculature by swollen fat-laden hepatocytes, and enhanced production of several cytokines. The beneficial effects of Wy-14,643 may be exerted by dampening adhesion molecule and cytokine responses, and activating NF-kappaB, IL-6 production, and p38 kinase to effect cell cycle entry.

Microvascular dysfunction in hepatic ischemia-reperfusion injury in pigs.

Although hepatic ischemia-reperfusion (I/R) injury has been investigated for more than two decades, histopathological documentation is limited. As a result, three pig livers with I/R injury and three control livers were injected with colored media, cut into 14 segments, and examined by light microscopy together with microscopic map making. In livers with I/R injury, lobules were identified as being occluded or unoccluded. The proportion of the occluded lobules increased in a caudocephalic fashion, while that of the unoccluded lobules decreased (chi(2) for linear trend, P<0.0001). Especially in the occluded lobules, swollen hepatic plates displayed various forms of cellular distortion. Collapsed sinusoids containing leukocyte aggregation and shrunken central veins were observed together with reduced caliber of the contiguous sublobular veins. Portal vein constriction with loosening of the surrounding stroma suggestive of edema and hepatic artery dilation were also seen. Isolated arterioles and transintimal vasal outlets of the hepatic vein's vasa venarum were dilated and frequently observed. In conclusion, I/R injury affected the liver parenchyma, the microvasculature, and its surrounding stroma. The heterogeneous distribution of occluded and unoccluded lobules is suggested due to the difference of vascular structure in various liver segments. The constrictive/obstructive changes in the portosinusoidal-hepatic vascular profile suggest a definite increase in resistance at presinusoidal, sinusoidal, and proximal postsinusoidal levels, resulting in an expansion of the arterial shunt circulation.

Prevention of hepatic fibrosis in a murine model of metabolic syndrome with nonalcoholic steatohepatitis.

The endocannabinoid pathway plays an important role in the regulation of appetite and body weight, hepatic lipid metabolism, and fibrosis. Blockade of the endocannabinoid receptor CB1 with SR141716 promotes weight loss, reduces hepatocyte fatty acid synthesis, and is antifibrotic. D-4F, an apolipoprotein A-1 mimetic with antioxidant properties, is currently in clinical trials for the treatment of atherosclerosis. C57BL/6J mice were fed a high-fat diet for 7 months, followed by a 2.5-month treatment with either SR141716 or D-4F. SR141716 markedly improved body weight, liver weight, serum transaminases, insulin resistance, hyperglycemia, hypercholesterolemia, hyperleptinemia, and oxidative stress, accompanied by the significant prevention of fibrosis progression. D-4F improved hypercholesterolemia and hyperleptinemia without improvement in body weight, steatohepatitis, insulin resistance, or oxidative stress, and yet, there was significant prevention of fibrosis. D-4F prevented culture-induced activation of stellate cells in vitro. In summary, C57BL/6J mice given a high-fat diet developed features of metabolic syndrome with nonalcoholic steatohepatitis and fibrosis. Both SR141716 and D-4F prevented progression of fibrosis after onset of steatohepatitis, ie, a situation comparable to a common clinical scenario, with D-4F seeming to have a more general antifibrotic effect. Either compound therefore has the potential to be of clinical benefit.

The hepatic microvascular system in health and its response to toxicants.

This review briefly summarizes what is known about the dynamic morphology of the hepatic microvascular system that includes all vessels in the liver with a diameter less than 300 microm and various morphological sites within these vessels that regulate the distribution of blood flow. The latter include the various segments of the afferent portal venules and hepatic arterioles, the sinusoids, and central and hepatic venules. Sinusoids are unique exchange vessels lined by fenestrated endothelial cells which have important endocytotic functions and phagocytic Kupffer cells which are important for host defense. These are encircled by extraluminal stellate cells that are specialized pericytes containing fat droplets that store vitamin A. The principle sites for regulating blood flow are in the sinusoidal network with stellate and endothelial cells playing a major role in regulating the diameters of sinusoids and the distribution of blood flow in individual sinusoids, lobules, or segments of lobules. The sinusoidal endothelial cells are a sensitive and early target for several toxicants. For example, as early as 30 minutes after the administration of acetaminophen, the endothelial cells become swollen and begin to lose the ability to endocytose ligands. Within 2 hr, gaps through the cytoplasm appear formed by the destruction and/or coalescence of fenestrae which permit red blood cells to penetrate into the space of Disse. Subsequently, the sinusoid may collapse or disintegrate reducing blood flow.

Old age and the hepatic sinusoid.

Morphological changes in the hepatic sinusoid with old age are increasingly recognized. These include thickening and defenestration of the liver sinusoidal endothelial cell, sporadic deposition of collagen and basal lamina in the extracellular space of Disse, and increased numbers of fat engorged, nonactivated stellate cells. In addition, there is endothelial up-regulation of von Willebrand factor and ICAM-1 with reduced expression of caveolin-1. These changes have been termed age-related pseudocapillarization. The effects of old age on Kupffer cells are inconsistent, but impaired responsiveness is likely. There are functional implications of these aging changes in the hepatic sinusoid. There is reduced sinusoidal perfusion, which will impair the hepatic clearance of highly extracted substrates. Blood clearance of a variety of waste macromolecules takes place in liver sinusoidal endothelial cells (LSECs). Previous studies indicated either that aging had no effect, or reduced the endocytic capacity of LSECs. However, a recent study in mice showed reduced endocytosis in pericentral regions of the liver lobules. Reduced endocytosis may increase systemic exposure to potential harmful waste macromolecules such as advanced glycation end products Loss of fenestrations leads to impaired transfer of lipoproteins from blood to hepatocytes. This provides a mechanism for impaired chylomicron remnant clearance and postprandial hyperlipidemia associated with old age. Given the extensive range of substrates metabolized by the liver, age-related changes in the hepatic sinusoid and microcirculation have important systemic implications for aging and age-related diseases.

Effects of old age on vascular complexity and dispersion of the hepatic sinusoidal network.

OBJECTIVES: In old age, there are marked changes in both the structure of the liver sinusoidal endothelial cell and liver perfusion. The objective of this study was to determine whether there are also aging changes in the microvascular architecture and vascular dispersion of the liver that might influence liver function. METHODS: Vascular corrosion casts and light micrographs of young (4 months) and old (24 months) rat livers were compared. Fractal and Fourier analyses and micro-computed tomography were used. Vascular dispersion was determined from the dispersion number for sucrose and 100-nm microspheres in impulse response experiments. RESULTS: Age did not affect sinusoidal dimensions, sinusoidal density, or dispersion number. There were changes in the geometry and complexity of the sinusoidal network as determined by fractal dimension and degree of anisotropy. CONCLUSIONS: There are small, age-related changes in the architecture of the liver sinusoidal network, which may influence hepatic function and reflect broader aging changes in the microcirculation. However, sinusoidal dimensions and hepatic vascular dispersion are not markedly influenced by old age.

Age-related changes in the liver sinusoidal endothelium: a mechanism for dyslipidemia.

The liver sinusoidal endothelial cell (LSEC) influences the transfer of substrates between the sinusoidal blood and hepatocytes and has a major role in endocytosis; therefore, changes in the LSEC have significant implications for hepatic function. There are major morphological changes in the LSEC in old age called pseudocapillarization. These changes include increased LSEC thickness and reduced numbers of pores in the LSEC, which are called fenestrations. Pseudocapillarization has been found in old humans, rats, mice, and nonhuman primates. In addition, old age is associated with impaired LSEC endocytosis and increased leukocyte adhesion, which contributes to reduced hepatic perfusion. Given that fenestrations in the endothelium allow passage of some lipoproteins, including chylomicron remnants, age-related reduction in fenestrations impairs hepatic lipoprotein metabolism. In old rats, caloric restriction was associated with complete preservation of LSEC morphology and fenestrations. In conclusion, pseudocapillarization of the LSEC is a newly discovered aging change that, through its effects on lipoproteins, contributes to the association between old age, dyslipidemia, and vascular disease.

Diannexin, a novel annexin V homodimer, provides prolonged protection against hepatic ischemia-reperfusion injury in mice.

BACKGROUND AND AIMS: Ischemia-reperfusion injury (IRI) remains an important cause of liver failure after hepatic surgery or transplantation. The mechanism seems to originate within the hepatic sinusoid, with damage to endothelial cells, an early, reproducible finding. Sinusoidal endothelial cells (SECs), damaged during reperfusion, activate and recruit inflammatory cells and platelets. We hypothesized that a recombinant human annexin V homodimer, Diannexin, would protect SECs from reperfusion injury. METHODS: We tested this proposal in a well-characterized in vivo murine partial hepatic IRI model. RESULTS: Whether administered 5 minutes or 24 hours before or 1 hour after ischemia-reperfusion, Diannexin (100-1000 microg/kg) almost completely protected against liver injury. The protective efficacy conferred by Diannexin was highly visible at the microcirculatory level. Thus, although IR in this model is associated with early swelling and gap formation in SECs, Diannexin ameliorated these effects as shown by >80% reduction in alanine aminotransferase values during the early phase of reperfusion injury (2 hours) and near normalization of liver necrosis and inflammation in the late phase of inflammatory recruitment (24 hours). Consistent with the proposed role of SEC injury in hepatic IRI, Diannexin also decreased hepatic expression of proinflammatory molecules (MIP-2, ICAM-1, VCAM), abolished leukocyte and platelet adherence to damaged SECs, and, by in vivo microscopy, Diannexin preserved microcirculatory blood flow and hepatocyte integrity during reperfusion. CONCLUSIONS: Diannexin is an apparently safe therapeutic protein that provides prolonged protection against hepatic IRI via cytoprotection of SECs, thereby interrupting secondary microcirculatory inflammation and coagulation.

Age-related changes in the hepatic microcirculation in mice.

Aging of the liver is associated with impaired metabolism of drugs, adverse drug interactions, and susceptibility to toxins. Since reduced hepatic blood flow is suspected to contribute this impairment, we examined age-related alterations in hepatic microcirculation. Livers of C57Bl/6 mice were examined at 0.8 (pre-pubertal), 3 (young adult), 14 (middle-aged), and 27 (senescent) months of age using in vivo and electron microscopic methods. The results demonstrated a 14% reduction in the numbers of perfused sinusoids between 0.8 and 27 month mice associated with 35% reduction in sinusoidal blood flow. This was accompanied by an inflammatory response evidenced by a fivefold increase in leukocyte adhesion in 27 month mice, up-regulated expression of ICAM-1, and increases in intrahepatic macrophages. Sinusoidal diameter decreased 6-10%. Liver sinusoidal endothelial cell (LSEC) dysfunction was seen as early as 14 months when there was a threefold increase in the numbers of swollen LSEC. The endocytotic capacity of LSEC also was found to be reduced in older animals. The sinusoidal endothelium in 27 month old mice exhibited pseudocapillarization. In conclusion, the results suggest that leukocyte accumulation in the sinusoids and narrowing of sinusoidal lumens due to pseudocapillarization and dysfunction of LSEC reduce sinusoidal blood flow in aged livers.

Reduced inflammatory response and increased microcirculatory disturbances during hepatic ischemia-reperfusion injury in steatotic livers of ob/ob mice.

Steatosis is a major risk factor for complications after liver surgery. Since neutrophil cytotoxicity is critical for ischemia-reperfusion injury in normal livers, the aim of the present study was to evaluate whether an exaggerated inflammatory response could cause the increased injury in steatotic livers. In C57Bl/6 mice, 60 min of warm hepatic ischemia triggered a gradual increase in hepatic neutrophil accumulation during reperfusion with peak levels of 100-fold over baseline at 12 h of reperfusion. Neutrophil extravasation and a specific neutrophil-induced oxidant stress (immunostaining for hypochlorous acid-modified epitopes) started at 6 h of reperfusion and peaked at 12-24 h. Ob/ob mice, which had a severe macrovesicular steatosis, suffered significantly higher injury (alanine transaminase activity: 18,000 +/- 2,100 U/l; 65% necrosis) compared with lean littermates (alanine transaminase activity: 4,900 +/- 720 U/l; 24% necrosis) at 6 h of reperfusion. However, 62% fewer neutrophils accumulated in steatotic livers. This correlated with an attenuated increase in mRNA levels of several proinflammatory genes in ob/ob mice during reperfusion. In contrast, sham-operated ob/ob mice had a 50% reduction in liver blood flow and 35% fewer functional sinusoids compared with lean littermates. These deficiencies in liver blood flow and the microcirculation were further aggravated only in ob/ob mice during reperfusion. The attenuated inflammatory response and reduced neutrophil-induced oxidant stress observed in steatotic livers during reperfusion cannot be responsible for the dramatically increased injury in ob/ob mice. In contrast, the aggravated injury appears to be mediated by ischemic necrosis due to massive impairment of blood and oxygen supply in the steatotic livers.

Substance P mediates cerulein-induced pancreatic microcirculatory dysfunction in mice.

OBJECTIVES: The present study was conducted to examine the contribution of substance P to the pancreatic microcirculatory dysfunction during acute pancreatitis. METHODS: Pancreatitis was elicited by up to 6 hourly injections of cerulein (50 microg/kg IP) in male C57Bl/6 mice. At 0, 1, 3, and 6 hours after cerulein treatment, the pancreatic microvasculature in anesthetized mice was studied using established high-resolution in vivo microscopic methods. RESULTS: Treatment of mice with cerulein for 6 hours caused a 30% decrease in capillary perfusion and the diameter of the capillaries and an increase in microvascular permeability (20%) and interstitial space (30-fold). The administration of the substance P receptor antagonist (D-Arg1, D-Pro2, D-Trp7,9, Leu11) (2 mg/kg IP) minimized the pancreatic microcirculatory dysfunction 3 hours after cerulein treatment. The superfusion of substance P for 0.5 hours decreased the diameter (by 22%) and increased microvascular permeability (by 23%) along with interstitial space (22-fold increase). Blockade of substance P receptor attenuated substance P-induced pancreatic microcirculatory dysfunction. CONCLUSIONS: These results suggest that substance P mediates pancreatic microcirculatory dysfunction during the development of acute pancreatitis.

Intestinal microcirculatory dysfunction during the development of experimental necrotizing enterocolitis.

The aim of this study was to evaluate changes in intestinal microcirculation during necrotizing enterocolitis (NEC) and to examine the effect of endothelin (ET)-1 on the intestinal microcirculation. Prematurely born rats were either hand-fed formula (NEC) or dam fed (DF) and were exposed to asphyxia and cold stress twice daily to induce disease. At 0, 2, 3, and 4 d after the birth, the microcirculation in the ileum was examined using in vivo microscopic methods. The nutritive microvascular perfusion in the NEC group was progressively compromised from d 3 to d 4 (35% and 50% decrease, respectively) when compared with DF rats. Concomitantly, intestinal blood flow assessed by laser Doppler flowmetry was significantly reduced at d 2, 3, and 4 (by 31%, 36%, and 73%, respectively). Levels of ET-1 mRNA in the ileum were increased 3.7-fold. Microvascular responses to topically applied ET-1 were significantly increased in the NEC group, which was associated with decreased expression of ETB receptor. These results suggest that microcirculatory dysfunction in the distal ileum of neonatal rats with NEC contributes to disease progression and that enhanced microvascular responsiveness to ET-1 may participate in these microcirculatory disturbances.