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.

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.

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.

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.

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.

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.

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.

Sinusoidal endothelial cells as an early target for hepatic toxicants.

Recent studies demonstrate that the hepatic sinusoidal endothelial cells (SEC) are a sensitive direct target for early toxicity to acetaminophen (paracetamol, APAP) and this toxicity is exacerbated following a single and multiple week-end type alcoholic binge(s). SEC become swollen and begin to lose the ability to endocytose FITC-FSA, a ligand for the scavenger receptor, as early as 30 minutes after the administration of APAP. Gaps through the SEC appear to be formed by the destruction and/or coalescence of fenestrae and are seen as early as 2 hrs after the administration of APAP which is prior to any evidence of injury to parenchymal cells. The gaps permit red blood cells to penetrate into the Space of Disse. Subsequently, the sinusoid may collapse or disintegrate reducing blood flow. The gaps are larger and more frequent in ethanol binged animals subsequently treated with APAP. Similar gaps are seen in the early stages of hepatic venoocclusive disease. Administration of a NO donor or a MMP-2 and MMP-9 inhibitor minimizes endothelial injury and red blood cell penetration into the Space of Disse. The injury is exacerbated when an inhibitor of eNOS is administered and minimized when iNOS is inhibited suggesting a protective role for constitutive NO derived from SEC. Both NO and MMPs are known to affect the cytoskeleton of SEC which in turn affects the formation and maintenance of the fenestrae.

Inhibition of matrix metalloproteinases minimizes hepatic microvascular injury in response to acetaminophen in mice.

The acetaminophen (APAP)-induced hepatic centrilobular necrosis is preceded by hepatic microcirculatory dysfunction including the infiltration of erythrocytes into the space of Disse. The purpose of this study was to examine the involvement of matrix metalloproteinases (MMPs) in the hepatic microvascular injury elicied by APAP. Male C57Bl/6 mice were pretreated with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid, an MMP-2/MMP-9 inhibitor (5 mg/kg, ip) 30 min before oral gavage with 600 mg/kg of APAP. The hepatic microvasculature in anesthetized mice was observed using established in vivo microscopic methods 2 and 6 h after APAP. The levels of mRNAs and activities of MMP-2 and MMP-9 in the liver were increased from 1 h through 6 h after APAP gavage. APAP increased alanine transferase (ALT) levels (41.1-fold) and resulted in centrilobular hemorrhagic necrosis at 6 h. Pretreatment with 2-[(4-biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated ALT values by 71% as well as the necrosis. APAP decreased the numbers of perfused sinusoids in centrilobular regions by 30% and increased the area occupied by infiltrated erythrocytes into Disse space. 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid restored the sinusoidal perfusion to 90% of control levels and minimized extrasinusoidal area occupied by erythrocytes. The present study showed that increased MMPs during APAP intoxication are associated with hepatocellular damage and with hepatic microcirculatory dysfunction including impaired sinusoidal perfusion and infiltration of erythrocytes in Disse space. 2-[(4-Biphenylsulfonyl) amino]-3-phenyl-propionic acid attenuated APAP-induced parenchymal and microvascular injury. These results suggest that MMPs participate in APAP hepatotoxicity mediated by sinusoidal endothelial cell injury, which results in impairment of microcirculation.

The importance of anatomy in health professions education and the shortage of qualified educators.

The current shortage of faculty qualified to teach anatomy in U.S. medical schools is reversible. Sufficient numbers of individuals are in the pipeline to provide a future cadre of well-trained faculty members educating students in gross anatomy. The challenge is to realign departmental, institutional, and federal training grant priorities and resources, creating incentives for graduate students, postdoctoral fellows, and faculty members to stay the course and become the teachers needed to educate the next generation of health professionals. These strategies include (but are not limited to) team-teaching gross anatomy, thereby distributing the time commitments of a laboratory-based course more widely within a department; funds made available from the administration of medical schools to allow postdoctoral fellows to participate in teaching and providing compensation for the research activities; using "mission-based budgeting" to specifically compensate for faculty teaching time; and, finally, re-instituting federally funded training grants that solved this same teaching crisis in the not-too-distant past.

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.

Enhanced green fluorescent protein-transfection of murine colon carcinoma cells: key for early tumor detection and quantification.

Many animal models for metastatic colorectal cancer represent clinical manifestations just inaccurately. We introduce a novel mouse model for metastastatic colorectal cancer. In order to remain close to the clinical disease a syngenic murine colon carcinoma cell line (colon 26 cells) was transfected with enhanced green fluorescent protein (EGFP). The transfected cells maintain the highly malignant attributes of the wild-type cells. Following injection into the portal circulation of Balb/c-mice, liver metastases occur in the same time span. Using the fluorescent attributes of the transfected cells, an approximation of the tumor load in liver tissue can be achieved by fluorescence activated cell sorting (FACS) and fluoroscan analysis. Tumor cell load in liver tissue can be accurately measured by Northern blot and Western blot analysis of liver tissue containing EGFP-transfected colon cancer metastases (1250 cells/mg liver tissue and 1000 cells/mg liver tissue) respectively. Confocal microscopy and intravital microscopy confirmed the growth of tumor metastases, originating from the intravascular compartments. The presented animal model using EGFP-transfected colon 26 cells allows the detecting of tumor growth in vivo and post mortem, as well as an accurate quantification of the tumor load in the liver tissue.

Effect of curcuminoids as anti-inflammatory agents on the hepatic microvascular response to endotoxin.

Curcuminoids, derived from the plant Curcuma domestica Val., have been shown to be free radical scavengers that suppress the production of superoxide by macrophages and potent anti-inflammatory agents that inhibit the lipopolysacharide (LPS)-induced production of tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, and the activation of nuclear factor (NF)-kappaB in human monocytic derived cells. The present study was undertaken to determine the efficacy of curcuminoids in inhibiting the hepatic microvascular inflammatory response elicited by LPS. BALB/C mice were gavaged intragastricly with curcuminoids [40 mg/kg body weight (bw) or 80 mg/kg bw] 1 h before intravenous injection of LPS (Escherichia coli, O111:B4, 100 microg/kg bw). The liver was examined 2 h after LPS injection using in vivo microscopic methods. LPS-treated mice showed significantly increased phagocytic activity of centrilobular Kupffer cells. The numbers of leukocytes adhering to the sinusoidal wall and swollen endothelial cells increased significantly in both the periportal and centrilobular regions, concomitant with a reduction in the numbers of sinusoids containing flow. Pretreatment with curcuminoids at the doses of 40 mg/kg bw or 80 mg/kg bw to endotoxemic mice significantly reduced the phagocytic activity of Kupffer cells, the numbers of adhering leukocytes and swollen endothelial cells. As a result, the number of sinusoids containing flow was increased in animals treated with 40 mg/kg curcuminoids and restored to control levels with 80 mg/kg curcuminoids. Neutrophil sequestration was reduced when measured in sections stained with naphtol AS-D chloroacetate esterase technique. These results demonstrate that curcuminoids are effective in suppressing the hepatic microvascular inflammatory response to LPS and may be a natural alternative anti-inflammatory substance.

Differential hepatotoxicity induced by cadmium in Fischer 344 and Sprague-Dawley rats.

A number of reports document that Fischer 344 (F344) rats are more susceptible to chemically induced liver injury than Sprague-Dawley (SD) rats. Cadmium (CdCl2), a hepatotoxicant that does not require bioactivation, was used to better define the biological events that are responsible for the differences in liver injury between F344 and SD rats. CdCl2 (3 mg/kg) produced hepatotoxicity in both rat strains, but the hepatic injury was 18-fold greater in F344 rats as assessed by plasma alanine aminotransferase (ALT) activity. This difference in toxicity was not observed when isolated hepatocytes were incubated with CdCl2 in vitro, indicating that other cell types contribute to Cd-induced hepatotoxicity in vivo. Indeed, the sieve plates of hepatic endothelial cells (EC) in F344 rats were damaged to a greater degree than EC in SD rats. Additionally, Kupffer cell (KC) inhibition reduced hepatotoxicity in both strains, suggesting that this cell type is involved in the progression of CdCl2-induced hepatotoxicity. Moreover, enhanced synthesis of heat shock protein 72 occurred earlier in the SD rat. Maximal levels of hepatic metallothionein (MT), a protein associated with cadmium tolerance, were greater in SD rats. These protective factors may limit CdCl2-induced hepatocellular injury in SD compared with F344 rats by reducing KC activation and the subsequent inflammatory response that allows for the progression of hepatic injury.

Anatomy of efferent hepatic nerves.

The role of neural elements in regulating blood flow through the hepatic sinusoids, solute exchange, and parenchymal function is incompletely understood. This is due in part to limited investigation in only a few species whose hepatic innervation may differ significantly from humans. For example, most experimental studies have used rats and mice having livers with little or no intralobular innervation. In contrast, most other mammals, including humans, have aminergic and peptidergic nerves extending from perivascular plexus in the portal space into the lobule, where they course in Disse's space in close relationship to stellate cells (fat storing cells of Ito) and hepatic parenchymal cells. While these fibers extend throughout the lobule, they predominate in the periportal region. Cholinergic innervation, however, appears to be restricted to structures in the portal space and immediately adjacent hepatic parenchymal cells. Neuropeptides have been colocalized with neurotransmitters in both adrenergic and cholinergic nerves. Neuropeptide Y (NPY) has been colocalized in aminergic nerves supplying all segments of the hepatic-portal venous and the hepatic arterial and biliary systems. Nerve fibers immunoreactive for substance P and somatostatin follow a similar distribution. Intralobular distribution of all of these nerve fibers is species-dependent and similar to that reported for aminergic fibers. Vasoactive intestinal peptide and calcitonin gene-related peptide (CGRP) are reported to coexist in cholinergic and sensory afferent nerves innervating portal veins and hepatic arteries and their branches, but not the other vascular segments or the bile ducts. Nitrergic nerves immunoreactive for neuronal nitric oxide (nNOS) are located in the portal tract where nNOS colocalizes with both NPY- and CGRP-containing fibers. In summary, the liver is innervated by aminergic, cholinergic, peptidergic, and nitrergic nerves. While innervation of structures in the portal tract is relatively similar between species, the extent and distribution of intralobular innervation are highly variable as well as species-dependent and may be inversely related to the density of gap junctions between contiguous hepatic parenchymal cells.

Hepatic microvascular development in relation to the morphogenesis of hepatocellular plates in neonatal rats.

The development of hepatic microvascular heterogeneity after birth, and its temporal relationship to the development of parenchymal cell plates have received little attention. As a result, the morphogenesis of some of the parameters contributing to this heterogeneity in suckling and weaned rats was studied as a function of time between postpartum days 4 and 30 using in vivo light microscopic, electron microscopic, and immunocytochemical methods. During the early suckling period, the sinusoid network is highly anastomotic, with little evidence of zonation, and the parenchymal cell plates contain multiple cells and are irregularly arranged throughout the lobule. Sinusoidal endothelial fenestration is sparse at 4 days, but phagocytic Kupffer cell (KC) function already exists and exhibits zonal heterogeneity, with more cells located in the periportal zone. With increasing age, endothelial fenestrae increase and organize as sieve plates. Widened centrilobular radial sinusoids form through a loss ("drop-out") of intersinusoidal sinusoids (ISS). Concomitantly, the associated cell plates straighten and become one cell thick. Hepatocyte DNA synthesis and mitosis are higher in the periportal zone, which retains thickened cell plates and anastomotic sinusoids. The centrilobular sinusoids may widen to accommodate the increased volume of blood that results from the loss of ISS as well as the increased numbers of periportal sinusoids containing flow that feed these vessels. KC phagocytic activity increases during the suckling period concomitant with an increase of gut-derived endotoxin in the portal blood, which suggests that the KCs may be releasing mediators that affect sinusoid diameter, blood flow, endothelial fenestration, and perhaps parenchymal growth either directly or through the stimulation of growth factors.

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.