Influence of microflow on hepatic sinusoid blood flow and red blood cell deformation

Hepatic sinusoids present complex anatomical structures such as the endothelial sieve pores and the Disse space, which govern the microscopic blood flow in the sinusoids and are associated with structural variations in liver fibrosis and cirrhosis. However, the contributions of the permeability of endothelial and collagen layers and the roughness of hepatocyte microvilli to the features of this microflow remain largely unknown. Here, an immersed boundary method coupled with a lattice Boltzmann method was adopted in an in vitro hepatic sinusoidal model, and flow field and erythrocyte deformation analyses were conducted by introducing three new source terms including permeability of the endothelial layer, resistance of hepatocyte microvilli and collagen layers, and deformation of red blood cells (RBCs). Numerical calculations indicated that alterations in endothelial permeability could significantly affect the flow velocity and flow rate distributions in hepatic sinusoids. Interestingly, a biphasic regulating pattern of shear stress occurred simultaneously on the surface of hepatocytes and the lower side of endothelium, i.e., the shear stress increased with increased thickness of hepatocyte microvilli and collagen layer when the endothelial permeability was high but decreased with the increase of the thickness at low endothelial permeability. Additionally, this specified microflow manipulates typical RBC deformation inside the sinusoid, yielding one-third of the variation of deformable index with varied endothelial permeability. These simulations not only are consistent with experimental measurements using in vitro liver sinusoidal chip but also elaborate the contributions of endothelial and collagen layer permeability and wall roughness. Thus, our results provide a basis for further characterizing this microflow and understanding its effects on cellular migration and deformation in the hepatic sinusoids.     

VEGFR1-Positive Macrophages Facilitate Liver Repair and Sinusoidal Reconstruction after Hepatic Ischemia/Reperfusion Injury

Liver repair after acute liver injury is characterized by hepatocyte proliferation, removal of necrotic tissue, and restoration of hepatocellular and hepatic microvascular architecture. Macrophage recruitment is essential for liver tissue repair and recovery from injury; however, the underlying mechanisms are unclear. Signaling through vascular endothelial growth factor receptor 1 (VEGFR1) is suggested to play a role in macrophage migration and angiogenesis. The aim of the present study was to examine the role of VEGFR1 in liver repair and sinusoidal reconstruction after hepatic ischemia/reperfusion (I/R). VEGFR1 tyrosine kinase knockout mice (VEGFR1 TK^-/- mice) and wild-type (WT) mice were subjected to hepatic warm I/R, and the processes of liver repair and sinusoidal reconstruction were examined. Compared with WT mice, VEGFR1 TK^-/- mice exhibited delayed liver repair after hepatic I/R. VEGFR1-expressing macrophages recruited to the injured liver showed reduced expression of epidermal growth factor (EGF). VEGFR1 TK^-/- mice also showed evidence of sustained sinusoidal functional and structural damage, and reduced expression of pro-angiogenic factors. Treatment of VEGFR1 TK^-/- mice with EGF attenuated hepatoceullar and sinusoidal injury during hepatic I/R. VEGFR1 TK^-/- bone marrow (BM) chimeric mice showed impaired liver repair and sinusoidal reconstruction, and reduced recruitment of VEGFR1-expressing macrophages to the injured liver. VEGFR1-macrophages recruited to the liver during hepatic I/R contribute to liver repair and sinusoidal reconstruction. VEGFR1 activation is a potential therapeutic strategy for promoting liver repair and sinusoidal restoration after acute liver injury.     

Evidence of species differences in the ultrastructure of the Hepatic sinusoid

Summary Hepatic sinusoids in the calf have been studied by light and electron microscopy. The endothelial lining is shown to be continuous and to be associated with a prominent basement membrane. The perisinusoidal space (of Disse) contains collagen fibrils and is otherwise nearly filled with hepatic cell microvilli. Perisinusoidal cells resembling fibroblasts or reticular cells in morphology and distribution are present in moderate numbers. Hepatic sinusoids in the rat were examined for purposes of comparison. In the rat the sinusoidal lining is discontinuous and there is no identifiable basement membrane. The perisinusoidal space is larger than in the calf and contains more sparsely distributed collagen fibrils. Perisinusoidal cells have not been identified. It is suggested that the difference in structure is not artifactitious but represents a species variation. This species difference seems important to record since observations on the rat and other small laboratory animals provide the basis for most of the current generalizations about the organization of the mammalian liver sinusoid. The functional significance of the two types of sinusoidal arrangement is not clear.A preliminary report of this work was presented at the VII th International Congress of Anatomists (Wood 1960).Supported in part by Grant H-2698 from the National Institutes of Health, U. S. Public Health Service.     

Cell surface changes and enzyme release during hypoxia and reoxygenation in the isolated, perfused rat liver

We examined the effects of hypoxia and reoxygenation in isolated, perfused rat livers. Hypoxia induced by a low rate of perfusion led to near anoxia confined to centrilobular regions of the liver lobule. Periportal regions remained normoxic. Within 15 min, anoxic centrilobular hepatocytes developed surface blebs that projected into sinusoids through endothelial fenestrations. Periportal hepatocytes were unaffected. Both scanning and transmission electron microscopy suggested that blebs developed by transformation of preexisting microvilli. Upon reoxygenation by restoration of a high rate of perfusion, blebs disappeared. Other changes included marked shrinkage of hepatocytes, enlargement of sinusoids, and dilation of sinusoidal fenestrations. There was also an abrupt increase in the release of lactate dehydrogenase and protein after reoxygenation, and cytoplasmic fragments corresponding in size and shape to blebs were recovered by filtration of the effluent perfusate. We also studied phalloidin and cytochalasin D, agents that disrupt the cytoskeleton. Both substances at micromolar concentrations caused rapid and profound alterations of cell surface topography. We conclude that hepatic tissue is quite vulnerable to hypoxic injury. The morphological expression of hypoxic injury seems mediated by changes in the cortical cytoskeleton. Reoxygenation causes disappearance of blebs and paradoxically causes disruption of cellular volume control and release of blebs as cytoplasmic fragments. Such cytoplasmic shedding provides a mechanism for selective release of hepatic enzymes by injured liver tissue.     

Scanning electron microscopy of normal rat liver: the surface structure of its cells and tissue components.

Scanning electron microscopy (SEM) allows the surface ultrastructure of intrahepatic cells and other tissue components of liver to be delineated. Excellent depth of focus of the SEM makes it possible to visualize surfaces of intact cells in their native configurations. This report details the surface characteristics and inter-relationships of hepatocytes and hepatic plates, sinusoidal endothelial cells and sinusoids, presumed Kupffer cells, vessels, bile ducts, connective tissue, and the capsule of rat liver. Hepatocytes present three structurally distinctive faces--the intercellular face containing flat surfaces and bile canaliculus, the sinusoidal face, and the connective tissue face which abuts portal tracts and hepatic veins. Sinusoidal endothelium is penetrated by large (1 to 3 mum) and small (0.1 mum) fenestrae, the latter occurring in clusters of up to 50. The width of bile canaliculi and distribution of large fenestrae vary proximodistally along hepatic plate or sinusoid. The cells of portal bile ductules contain microvilli located in linear rows and sparse cilia. Endothelium of hepatic artery and of portal vein is sparsely fenestrated.     

Endotoxin-induced liver hypoxia: defective oxygen delivery versus oxygen consumption.

In vivo EPR was used to investigate liver oxygenation in a hemodynamic model of septic shock in mice. Oxygen-sensitive material was introduced either (i) as a slurry of fine particles which localized at the liver sinusoids (pO2 = 44.39 +/- 5.13 mmHg) or (ii) as larger particles implanted directly into liver tissue to measure average pO2 across the lobule (pO2 = 4.56 +/- 1.28 mmHg). Endotoxin caused decreases in pO2 at both sites early (5-15 min) and at late time points (6 h after endotoxin; sinusoid = 11.22 +/- 2.48 mmHg; lobule = 1.16 +/- 0.42 mmHg). The overall pO2 changes observed were similar (74.56% versus 74.72%, respectively). Blood pressures decreased transiently between 5 and 15 min (12.88 +/- 8% decrease) and severely at 6 h (59 +/- 9% decrease) following endotoxin, despite volume replacement with saline. Liver and circulatory nitric oxide was elevated at these times. Liver oxygen extraction decreased from 44% in controls to only 15% following endotoxin, despite severe liver hypoxia. Arterial oxygen saturation, blood flow (hepatic artery), and cardiac output were unaffected. Pretreatment with l-NMMA failed to improve endotoxin-induced oxygen defects at either site, whereas interleukin-13 preserved oxygenation. These site-specific measurements of pO2 provide in vivo evidence that the principal cause of liver hypoxia during hypodynamic sepsis is reduced oxygen supply to the sinusoid and can be alleviated by maintaining sinusoidal perfusion.     

In vivo imaging of malaria parasites in the murine liver

The form of the malaria parasite inoculated by the mosquito, called the sporozoite, transforms inside the host liver into thousands of a new form of the parasite, called the merozoite, which infects erythrocytes. We present here a protocol to visualize in vivo the behavior of Plasmodium berghei parasites in the hepatic tissue of the murine host. The use of GFP-expressing parasites and a high-speed spinning disk confocal microscope allows for the acquisition of four-dimensional images, which provide a time lapse view of parasite displacement and development in tissue volumes. These data can be analyzed to give information on the early events of sporozoite penetration of the hepatic tissue, that is, sporozoite gliding in the liver sinusoids, crossing the sinusoidal barrier, gliding in the parenchyma and traversal of hepatocytes, and invasion of a final hepatocyte, as well as the terminal events of merosome and merozoite release from infected hepatocytes. Combined with the use of mice expressing fluorescent cell types or cell markers, the system will provide useful information not only on the primary infection process, but also on parasite interactions with the host immune cells in the liver.     

Hepatic elimination of flowing substrates: the distributed model

An earlier model of hepatic elimination with functionally identical sinusoids is extended by introducing statistical distributions of enzyme contents per sinusoid and of blood flow per sinusoid, these being either uncorrelated or closely correlated. The steady-state theory of the resulting distributed model is developed, including methods of determining experimentally the coefficients of variation of the distributions. Such determinations are made on an illustrative experimental example. Quantitative predictions of expected effects of changes in blood flow are given, including one for which the undistributed model predicts a null effect. Shapes of the postulated distributions are discussed only in relation to observable effects. Effects of the distributions are compared with maximum possible effects of incomplete equilibration of substrate within each sinusoidal cross-section, and methods for distinguishing these effects from each other are outlined.     

Microcirculation in the murine liver: a computational fluid dynamic model based on 3D reconstruction from in vivo microscopy

The liver is organized in hexagonal functional units – termed lobules – characterized by a rather peculiar blood microcirculation, due to the presence of a tangled network of capillaries – termed sinusoids. A better understanding of the hemodynamics that governs liver microcirculation is relevant to clinical and biological studies aimed at improving our management of liver diseases and transplantation.Herein, we built a CFD model of a 3D sinusoidal network, based on in vivo images of a physiological mouse liver obtained with a 2-photon microscope. The CFD model was developed with Fluent 16.0 (ANSYS Inc., Canonsburg, PA), particular care was taken in imposing the correct boundary conditions representing a physiological state. To account for the remaining branches of the sinusoids, a lumped parameter model was used to prescribe the correct pressure at each outlet. The effect of an adhered cell on local hemodynamics is also investigated for different occlusion degrees.The model here proposed accurately reproduces the fluid dynamics in a portion of the sinusoidal network in mouse liver. Mean velocities and mass flow rates are in agreement with literature values from in vivo measurements. Our approach provides details on local phenomena, hardly described by other computational studies, either focused on the macroscopic hepatic vasculature or based on homogeneous porous medium model.     

Effect of the nitric oxide donor V-PYRRO/NO on portal pressure and sinusoidal dynamics in normal and cirrhotic mice

Reduced sinusoidal endothelial nitric oxide (NO) production contributes to increased intrahepatic resistance and portal hypertension after liver injury. We hypothesized that V-PYRRO/NO, an NO donor prodrug metabolized "specifically" in the liver, would reduce portal venous pressure (PVP) without affecting the systemic vasculature. Liver injury was induced in male BALB/c mice by weekly CCl(4) gavage. PVP and mean arterial pressure were recorded during intravenous administration of V-PYRRO/NO. In vivo microscopy was used to monitor sinusoidal diameter and flow during drug administration. Mean PVP was increased in CCl(4)-treated mice compared with sham-treated mice. In dose-response experiments, the minimum dose of PYRRO/NO required to acutely lower PVP by 20%, the amount believed to yield a clinically meaningful outcome, was 200 nmol/kg. This dose decreased portal pressure in cirrhotic (23.4 +/- 2.0%, P < 0.001 vs. vehicle) and sham-treated (19.5 +/- 2.3%, P < 0.001 vs. vehicle) animals by a similar magnitude. This concentration also led to dilation of hepatic sinusoids and an increase in sinusoidal volumetric flow, consistent with a reduction of intrahepatic resistance. The effect of V-PYRRO/NO on mean arterial pressure was significant at all concentrations tested, including the lowest, 30 nmol/kg (P < 0.001 vs. vehicle for all doses). We conclude that V-PYRRO/NO had widespread vascular effects and, as such, is unlikely to be suitable for treatment of portal hypertension. As the potential of this or other similar compounds for treatment of portal hypertension is evaluated, effects on the systemic vasculature will also need to be considered.     

Analysis of the sinusoidal endothelial cell organization during the developmental stage of the fetal rat liver using a sinusoidal endothelial cell specific antibody, SE-1

The sinusoid organization during the development of fetal rat livers was studied using a SE-1 antibody, which we have previously established as a specific monoclonal antibody against rat sinusoidal endothelial cell (SEC). Expression and localization of the SE-1 antigen in the liver tissues of 13- to 21-day-old fetuses were immunofluorescently and immunoelectron microscopically examined. The first positive fluorescence was observed in the immature liver of 15-day-old fetuses. The initial positive staining was randomly distributed in the liver parenchyma and showed no direct relation to the large vessels which may be derived from the fetal vitelline veins. The positive linear staining increased in number and connected with each other during the course of development. The SE-1 staining pattern and the sinusoidal arrangement became similar to those of the adult liver after 20th day of gestation. Immunoelectron microscopically, the immature SEC showed a weak positive reaction for the SE-1 antigen at their membrane and was observed together with immature hepatocytes and hematopoietic cells in the 15-day-old fetal liver. Along with the liver development, SEC formed a sinusoid structure closely associated with hepatocytes and came to strongly express the SE-1 antigen. These results indicate that the organization of the hepatic sinusoid may start at around 15th day of the gestation and occurs randomly in the fetal liver parenchyma. It is also suggested that the expression of SE-1 antigen is possibly regulated by the intimate association with hepatocytes.     

Intravascular Immune Surveillance by CXCR6+ NKT Cells Patrolling Liver Sinusoids

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6⁺ cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.     

Intravascular immune surveillance by CXCR6+ NKT cells patrolling liver sinusoids

We examined the in vivo behavior of liver natural killer T cells (NKT cells) by intravital fluorescence microscopic imaging of mice in which a green fluorescent protein cDNA was used to replace the gene encoding the chemokine receptor CXCR6. NKT cells, which account for most CXCR6⁺ cells in liver, were found to crawl within hepatic sinusoids at 10–20 μm/min and to stop upon T cell antigen receptor activation. CXCR6-deficient mice exhibited a selective and severe reduction of CD1d-reactive NKT cells in the liver and decreased susceptibility to T-cell-dependent hepatitis. CXCL16, the cell surface ligand for CXCR6, is expressed on sinusoidal endothelial cells, and CXCR6 deficiency resulted in reduced survival, but not in altered speed or pattern of patrolling of NKT cells. Thus, NKT cells patrol liver sinusoids to provide intravascular immune surveillance, and CXCR6 contributes to liver-based immune responses by regulating their abundance.     

Transient expression of laminin alpha1 chain in regenerating murine liver: restricted localization of laminin chains and nidogen-1

Most interstitia between epithelial and endothelial cells contain basal laminae (BLs), as defined by electron microscopy. However, in liver, the sinusoidal interstitium (called space of Disse) between hepatocytes and sinusoidal endothelial cells (SECs) lacks BLs. Because laminins are major components of BLs throughout the body, whether laminins exist in sinusoids has been a controversial issue. Despite recent advances, the distribution and expression of laminin chains have not been well defined in mammalian liver. Here, using a panel of antibodies, we examined laminins in normal and regenerating mouse livers. Of alpha chains, alpha5 was widely observed in all BLs except for sinusoids, while the other alpha chains were variously expressed in Glisson's sheath and central veins. Laminin gamma1 was also distributed to all BLs except for sinusoids. Although the beta2 chain was observed in all BLs and sinusoids, the expression of beta1 chain was restricted to Glisson's sheath. Detailed analysis of regenerating liver revealed that alpha1 and gamma1 chains appeared in sinusoids and were produced by stellate cells. The staining of alpha1 and gamma1 chains reached its maximum intensity at 6 days after two-thirds partial hepatectomy (PHx). Moreover, in vitro studies showed that alpha1-containing laminin promoted spreading of sinusoidal endothelial cells (SECs) isolated from normal liver, but not other hepatic cells. In addition, SECs isolated from regenerating liver elongated pseudopodia on alpha1-containing laminin more so than did cells from normal liver. The transient expression of laminin alpha1 may promote formation of sinusoids after PHx.     

Numerical simulation of flow characteristics in a permeable liver sinusoid with leukocytes

Double-layered channels of sinusoid lumen and Disse space separated by fenestrated liver sinusoidal endothelial cells (LSECs) endow the unique mechanical environment of the liver sinusoid network, which further guarantees its biological function. It is also known that this mechanical environment changes dramatically under liver fibrosis and cirrhosis, including the reduced plasma penetration and metabolite exchange between the two flow channels and the reduced Disse space deformability. The squeezing of leukocytes through narrow sinusoid lumen also affects the mechanical environment of liver sinusoid. To date, the detailed flow-field profile of liver sinusoid is still far from clear due to experimental limitations. It also remains elusive whether and how the varied physical properties of the pathological liver sinusoid regulate the fluid flow characteristics. Here a numerical model based on the immersed boundary method was established, and the effects of Disse space and leukocyte elasticities, endothelium permeability, and sinusoidal stenosis degree on fluid flow as well as leukocyte trafficking were specified upon a mimic liver sinusoid structure. Results showed that endothelium permeability dominantly controlled the plasma penetration velocity across the endothelium, whereas leukocyte squeezing promoted local penetration and significantly regulated wall shear stress on hepatocytes, which was strongly related to the Disse space and leukocyte deformability. Permeability and elasticity cooperatively regulated the process of leukocytes trafficking through the liver sinusoid, especially for stiffer leukocytes. This study will offer new insights into deeper understanding of the elaborate mechanical features of liver sinusoid and corresponding biological function.     

Intrahepatic microvascular changes in carbon tetrachloride-induced cirrhotic livers in the rat.

Liver cirrhosis was produced in the rat by combined carbon tetrachloride-phenobarbitone treatment, and the microcirculation in the cirrhotic liver was observed by a quantitative in vivo transillumination technique. The total sinusoidal flow in the observed region of the cirrhotic liver did not differ significantly from that in the normal liver, despite the reduced number of sinusoids and the increased portal venous pressure. The cirrhotic liver also presented a fast-velocity population of portal and hepatic venules and sinusoids in addition to the normal slow-velocity population. The possible mechanism of these "arterialized" microvessels is discussed.     

Content of dolichol and retinol in isolated rat non-parenchymal liver cells

The liver sinusoids, that are considered as a functional unit, harbour four types of sinusoidal cells (Ito, Kupffer, endothelial and pit cells). Dolichol content has been determined in many tissues and subcellular compartments, alteration has been reported in many types of liver injury, but until now no data are available on its content in every type of sinusoidal non-parenchymal liver cells. Dolichol and retinol metabolism might intersect in their traffic in biological membranes. Intercellular as well as intracellular exchange of retinoids is an essential element of important processes occurring in liver cells. It has been suggested that the role of dolichol, besides being a carrier of oligosaccharides in the biosynthesis of N-linked glycoproteins, may be to modify membrane fluidity and permeability, and facilitate fusion of membranes. Dolichol in the membrane is intercalated between the two halves of the phospholipid bilayer, but its exact disposition is not known and the movement and distribution of retinoid in membranes may vary with the geometry of the membranes. Therefore the aim of this study is to obtain a global understanding of the sinusoidal system regarding dolichol and retinol content in each type of isolated rat liver sinusoidal cell, in normal conditions and after vitamin A administration. The information that can be drawn from the present results is that with normal vitamin A status of the animal, the dolichol content is almost uniform in all liver cells. After vitamin A supplementation, a great increase of dolichol, together with the known increase of retinol, can be measured only in a subpopulation of the Ito cells, the Ito-1 subfraction. Therefore in the cells that are present in the hepatic sinusoid, different pools of dolichol may have separate functions. Because retinol traffic among cells, membranes and plasma still remains to be fully understood, roles of dolichol in the exchange of vitamin A among sinusoidal liver cells are discussed. © 1998 John Wiley & Sons, Ltd.     

Anti-CD95-induced lethality requires radioresistant Fcgamma RII+ cells. A novel mechanism for fulminant hepatic failure

The Jo2 anti-mouse CD95 monoclonal antibody induces lethality in mice characterized by hepatocyte death and liver hemorrhage. Mice bearing a defect in Fas expression or in the Fas-mediated apoptotic pathway are resistant to Jo2. Here we show that FcgammaRII knockout mice or mice with monoclonal antibody-blocked FcgammaRII are also resistant to Jo2. The critical FcgammaRII(+) cells are radioresistant and could not be reconstituted with splenic cells. Death of sinusoidal lining cells and destruction of sinusoids were observed, consistent with the characteristic liver hemorrhage and the selective FcgammaRII expression in sinusoidal lining cells but not hepatocytes. Hemorrhage developed coincident with hepatocyte death and the sharp rise of serum alanine aminotransferase and alanine aminotransferase. Invariably, moribund mice showed severe liver hemorrhage and destruction of sinusoids. The data demonstrate a novel mechanism by which the destruction of liver sinusoids, induced by the Jo2-mediated co-engagement of Fas and FcgammaRII, leads to severe hemorrhage and lethal fulminant hepatitis.