Developing in vitro engineered hepatic tissues that exhibit stable phenotype is a major challenge in the field of hepatic tissue engineering. However, the rapid dedifferentiation of hepatic parenchymal (hepatocytes) and non-parenchymal (liver sinusoidal endothelial, LSEC) cell types when removed from their natural environment in vivo remains a major obstacle. The primary goal of this study was to demonstrate that hepatic cells cultured in layered architectures could preserve or potentially enhance liver-specific behavior of both cell types. Primary rat hepatocytes and rat LSECs (rLSECs) were cultured in a layered three-dimensional (3D) configuration. The cell layers were separated by a chitosan-hyaluronic acid polyelectrolyte multilayer (PEM), which served to mimic the Space of Disse. Hepatocytes and rLSECs exhibited several key phenotypic characteristics over a twelve day culture period. Immunostaining for the sinusoidal endothelial 1 antibody (SE-1) demonstrated that rLSECs cultured in the 3D hepatic model maintained this unique feature over twelve days. In contrast, rLSECs cultured in monolayers lost their phenotype within three days. The unique stratified structure of the 3D culture resulted in enhanced heterotypic cell-cell interactions, which led to improvements in hepatocyte functions. Albumin production increased three to six fold in the rLSEC-PEM-Hepatocyte cultures. Only rLSEC-PEM-Hepatocyte cultures exhibited increasing CYP1A1/2 and CYP3A activity. Well-defined bile canaliculi were observed only in the rLSEC-PEM-Hepatocyte cultures. Together, these data suggest that rLSEC-PEM-Hepatocyte cultures are highly suitable models to monitor the transformation of toxins in the liver and their transport out of this organ. In summary, these results indicate that the layered rLSEC-PEM-hepatocyte model, which recapitulates key features of hepatic sinusoids, is a potentially powerful medium for obtaining comprehensive knowledge on liver metabolism, detoxification and signaling pathways in vitro.
Liver sinusoidal endothelial cells (LSECs) form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII). Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene (uPA-NOG mice). Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.
It is increasingly appreciated that since cell and tissue functions are regulated by chemomechanical stimuli, precise control over such stimuli will improve the functionality of tissue models. However, due to the inherent difficulty in decoupling these cues as presented by extracellular materials, few studies have explored the independent modulation of biochemical and mechanical stimuli towards the manipulation of sustained cellular processes. Here, we demonstrate that both mechanical compliance and ligand presentation of synthetic, weak polyelectrolyte multilayers (PEMs) can be tuned independently to influence the adhesion and liver-specific functions of primary rat hepatocytes over extended in vitro culture (two weeks). These synthetic PEMs exhibited elastic moduli E ranging over 200 kPa -< E < 142 MPa, as much as one thousand-fold more compliant than tissue-culture polystyrene (E ∼ 2.5 GPa). The most compliant of these PEM substrata promoted hepatocyte adhesion and spheroidal morphology. Subsequent modification of PEMs with type I collagen and the proteoglycan decorin did not alter substrata compliance, but enhanced the retention of spheroids on surfaces and stabilized hepatic functions (albumin and urea secretion, CYP450 detoxification activity). Decorin exhibited unique compliance-mediated effects on hepatic functions, down-regulating the hepatocyte phenotype when presented on highly compliant substrata while up-regulating hepatocyte functions when presented on increasingly stiffer substrata. These results show that phenotypic functions of liver models can be modulated by leveraging synthetic polymers to study and optimize the interplay of biochemical and mechanical cues at the cell–material interface. More broadly, these results suggest an enabling approach for the systematic design of functional tissue models applied to drug screening, cell-based therapies and fundamental studies in development, physiology and disease.
Hepatocyte; Polyelectrolyte multilayers; Compliance; Surface modification; Chemomechanics
Liver Sinusoidal Endothelial Cells (LSEC) differ, both structurally and functionally, from endothelial cells (EC) lining blood vessels of other tissues. For example, in contrast to other EC, LSEC posses fenestrations, have low detectable levels of PECAM-1 expression, and in rat tissue, they distinctively express a cell surface marker recognized by the SE-1 antibody. These unique phenotypic characteristics seen in hepatic tissue are lost over time upon culture in vitro; therefore, this study sought to systematically examine the effects of microenvironmental stimuli, namely, extracellular matrix (ECM) and neighboring cells, on the LSEC phenotype in vitro. In probing the role of the underlying extracellular matrix, we identified collagen I and collagen III as well as mixtures of collagen I/collagen IV/fibronectin as having a positive effect on LSEC survival. Furthermore, using a stable hepatocellular model (hepatocyte-fibroblast) we were able to prolong the expression of both SE-1 and phenotypic functions of LSEC such as Factor VIII activity in co-cultured LSECs through the production of short-range paracrine signals. In the course of these experiments, we identified the antigen recognized by SE-1 as CD32b. Collectively, this study has identified several microenvironmental regulators of liver sinusoidal endothelial cells that prolong their phenotypic functions for up to 2 weeks in culture, enabling the development of better in vitro models of liver physiology and disease.
endothelial phenotype; SE-1; CD32b; extracellular matrix; hepatocytes
The ability of the liver to regenerate is crucial to protect liver function after injury and during chronic disease. Increases in hepatocyte growth factor (HGF) in liver sinusoidal endothelial cells (LSECs) are thought to drive liver regeneration. However, in contrast to endothelial progenitor cells, mature LSECs express little HGF. Therefore, we sought to establish in rats whether liver injury causes BM LSEC progenitor cells to engraft in the liver and provide increased levels of HGF and to examine the relative contribution of resident and BM LSEC progenitors. LSEC label-retaining cells and progenitors were identified in liver and LSEC progenitors in BM. BM LSEC progenitors did not contribute to normal LSEC turnover in the liver. However, after partial hepatectomy, BM LSEC progenitor proliferation and mobilization to the circulation doubled. In the liver, one-quarter of the LSECs were BM derived, and BM LSEC progenitors differentiated into fenestrated LSECs. When irradiated rats underwent partial hepatectomy, liver regeneration was compromised, but infusion of LSEC progenitors rescued the defect. Further analysis revealed that BM LSEC progenitors expressed substantially more HGF and were more proliferative than resident LSEC progenitors after partial hepatectomy. Resident LSEC progenitors within their niche may play a smaller role in recovery from partial hepatectomy than BM LSEC progenitors, but, when infused after injury, these progenitors engrafted and expanded markedly over a 2-month period. In conclusion, LSEC progenitor cells are present in liver and BM, and recruitment of BM LSEC progenitors is necessary for normal liver regeneration.
The liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC.
Endocytosis of five 125I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells.
Pig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC.
The layer-by-layer assembly of sequentially adsorbed, alternating polyelectrolytes has become increasingly important over the past two decades. The ease and versatility in assembling polyelectrolyte multilayers (PEMs) has resulted in numerous wide ranging applications of these materials. More recently, PEMs are being used in biological applications ranging from biomaterials, tissue engineering, regenerative medicine, and drug delivery. The ability to manipulate the chemical, physical, surface, and topographical properties of these multilayer architectures by simply changing the pH, ionic strength, thickness, and postassembly modifications render them highly suitable to probe the effects of external stimuli on cellular responsiveness. In the field of regenerative medicine, the ability to sequester growth factors and to tether peptides to PEMs has been exploited to direct the lineage of progenitor cells and to subsequently maintain a desired phenotype. Additional novel applications include the use of PEMs in the assembly of three-dimensional layered architectures and as coatings for individual cells to deliver tunable payloads of drugs or bioactive molecules. This review focuses on literature related to the modulation of chemical and physical properties of PEMs for tissue engineering applications and recent research efforts in maintaining and directing cellular phenotype in stem cell differentiation.
We evaluated the kinetics by which rat liver sinusoidal endothelial cells (LSECs) are repopulated in the reperfused transplanted liver after 18 hours of cold ischemic storage. We found that the majority of LSECs in livers cold-stored for 18 hours in University of Wisconsin solution are seriously compromised and often are retracted before transplantation. Sinusoids rapidly re-endothelialize within 48 hours of transplantation, and repopulation is coincident with up-regulation of hepatocyte vascular endothelial growth factor expression and vascular endothelial growth factor receptor-2 expression on large vessel endothelial cells and repopulating LSECs. Although re-endothelialization occurs rapidly, we show here, using several high-resolution imaging techniques and 2 different rat liver transplantation models, that engraftment of bone marrow–derived cells into functioning LSECs is routinely between 1% and 5%.
Bone marrow plays a measurable but surprisingly limited role in the rapid repopulation and functional engraftment of bone marrow–derived LSECs after cold ischemia and warm reperfusion.
Liver sinusoidal endothelial cells (LSECs) play an essential role in systemic waste clearance by effective endocytosis of blood-borne waste macromolecules. We aimed to study LSECs’ scavenger function during aging, and whether age-related morphological changes (eg, defenestration) affect this function, in F344/BN F1 rats. Endocytosis of the scavenger receptor ligand formaldehyde-treated serum albumin was significantly reduced in LSECs from old rats. Ligand degradation, LSEC protein expression of the major scavenger receptors for formaldehyde-treated serum albumin endocytosis, stabilin-1 and stabilin-2, and their staining patterns along liver sinusoids, was similar at young and old age, suggesting that other parts of the endocytic machinery are affected by aging. Formaldehyde-treated serum albumin uptake per cell, and cell porosity evaluated by electron microscopy, was not correlated, indicating that LSEC defenestration is not linked to impaired endocytosis. We report a significantly reduced LSEC endocytic capacity at old age, which may be especially important in situations with increased circulatory waste loads.
Aging; Hepatic sinusoid; Porosity; Stabilin; Scavenger endothelial cells
Stromal-derived factor (SDF)-1 is the main regulating factor for trafficking/homing of hematopoietic stem cells (HSC) to the bone marrow (BM). It is possible that this chemokine may also play a fundamental role in regulating the migration of HSC to several organs during extramedullary hematopoiesis. Because liver sinusoidal endothelial cells (LSEC) constitute an extramedullary niche for HSC, it is possible that these cells represent one of the main cellular sources of SDF-1 at the liver. Here, we show that LSEC express SDF-1 at the mRNA and protein level. Biological assays showed that conditioned medium from LSEC (LSEC-CM) stimulated the migration of BM progenitor lineage-negative (BM/Lin−) cells. This effect was significantly reduced by AMD3100, indicating that the SDF-1/CXCR4 axis is involved in the stimulatory migrating effect induced by LSEC-CM. Early localization of HSC in SDF-1–expressing LSEC microenvironment together with increased levels of this chemokine in hepatic homogenates was found in an experimental model of liver extramedullary hematopoiesis. Flow cytometry studies showed that LSEC express the CXCR4 receptor. Functional assays showed that activation of this receptor by SDF-1 stimulated the migration of LSEC and increased the expression of PECAM-1. Our findings suggest that LSEC through the production of SDF-1 may constitute a fundamental niche for regulation of HSC migration to the liver. To our knowledge, this is the first report showing that LSEC not only express and secrete SDF-1, but also its receptor CXCR4.
The IL-33/ST2 axis is known to be involved in liver pathologies. Although, the IL-33 levels increased in sera of viral hepatitis patients in human, the cellular sources of IL-33 in viral hepatitis remained obscure. Therefore, we aimed to investigate the expression of IL-33 in murine fulminant hepatitis induced by a Toll like receptor (TLR3) viral mimetic, poly(I:C) or by pathogenic mouse hepatitis virus (L2-MHV3). The administration of poly(I:C) plus D-galactosamine (D-GalN) in mice led to acute liver injury associated with the induction of IL-33 expression in liver sinusoidal endothelial cells (LSEC) and vascular endothelial cells (VEC), while the administration of poly(I:C) alone led to hepatocyte specific IL-33 expression in addition to vascular IL-33 expression. The hepatocyte-specific IL-33 expression was down-regulated in NK-depleted poly(I:C) treated mice suggesting a partial regulation of IL-33 by NK cells. The CD1d KO (NKT deficient) mice showed hepatoprotection against poly(I:C)-induced hepatitis in association with increased number of IL-33 expressing hepatocytes in CD1d KO mice than WT controls. These results suggest that hepatocyte-specific IL-33 expression in poly(I:C) induced liver injury was partially dependent of NK cells and with limited role of NKT cells. In parallel, the L2-MHV3 infection in mice induced fulminant hepatitis associated with up-regulated IL-33 expression as well as pro-inflammatory cytokine microenvironment in liver. The LSEC and VEC expressed inducible expression of IL-33 following L2-MHV3 infection but the hepatocyte-specific IL-33 expression was only evident between 24 to 32h of post infection. In conclusion, the alarmin cytokine IL-33 was over-expressed during fulminant hepatitis in mice with LSEC, VEC and hepatocytes as potential sources of IL-33.
The liver removes quickly the great bulk of virus circulating in blood, leaving only a small fraction to infect the host, in a manner characteristic of each virus. The scavenger cells of the liver sinusoids are implicated, but the mechanism is entirely unknown. Here we show, borrowing a mouse model of adenovirus clearance, that nearly all infused adenovirus is cleared by the liver sinusoidal endothelial cell (LSEC). Using refined immunofluorescence microscopy techniques for distinguishing macrophages and endothelial cells in fixed liver, and identifying virus by two distinct physicochemical methods, we localized adenovirus 1 minute after infusion mainly to the LSEC (∼90%), finding ∼10% with Kupffer cells (KC) and none with hepatocytes. Electron microscopy confirmed our results. In contrast with much prior work claiming the main scavenger to be the KC, our results locate the clearance mechanism to the LSEC and identify this cell as a key site of antiviral activity.
The liver has long been known as the garbage dump of the body, capable of rapidly removing hazardous pathogens and useless particles from the blood stream, thereby protecting the host. The only cell doing the removal has been thought to be the liver's macrophages. This is likely true for larger particles such as bacteria. But for smaller particles the size of virus or small antibody-antigen complexes, macrophages are probably not the cell responsible for the bulk of removal. We suggest, rather, it is the endothelial cell of the liver's blood circulatory system that takes up and destroys the majority of virus, doing so quickly (minutes) and extensively (>90%), leaving only a small fraction of circulating virus to infect the body in ways peculiar to each virus. To test this possibility, we infused mice intravenously with a harmless common cold virus and tracked its destination by molecular and microscopy methods. Affirming our conjecture, we found that ∼90% of the infused virus homed to the endothelium of the liver and ∼10% went to its macrophages. These data support a unique role, generally underappreciated, for the liver endothelium in viral clearance.
The normal liver is characterized by immunologic tolerance. Primary mediators of hepatic immune tolerance are liver sinusoidal endothelial cells (LSECs). LSECs block adaptive immunogenic responses to Ag and induce the generation of T regulatory cells. Hepatic fibrosis is characterized by both intense intrahepatic inflammation and altered hepatic immunity. We postulated that, in liver fibrosis, a reversal of LSEC function from tolerogenic to proinflammatory and immunogenic may contribute to both the heightened inflammatory milieu and altered intrahepatic immunity. We found that, after fibrotic liver injury from hepatotoxins, LSECs become highly proinflammatory and secrete an array of cytokines and chemokines. In addition, LSECs gain enhanced capacity to capture Ag and induce T cell proliferation. Similarly, unlike LSECs in normal livers, in fibrosis, LSECs do not veto dendritic cell priming of T cells. Furthermore, whereas in normal livers, LSECs are active in the generation of T regulatory cells, in hepatic fibrosis LSECs induce an immunogenic T cell phenotype capable of enhancing endogenous CTLs and generating potent de novo CTL responses. Moreover, depletion of LSECs from fibrotic liver cultures mitigates the proinflammatory milieu characteristic of hepatic fibrosis. Our findings offer a critical understanding of the role of LSECs in modulating intrahepatic immunity and inflammation in fibro-inflammatory liver disease.
Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs) using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ), i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ) transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.
DNA nanostructures have been attractive due to their structural properties resulting in many important breakthroughs especially in controlled assemblies and many biological applications. Here, we report a unique energy storage device which is a supercapacitor that uses nanostructured DNA hydrogel (Dgel) as a template and layer-by-layer (LBL)-deposited polyelectrolyte multilayers (PEMs) as conductors. Our device, named as PEM-Dgel supercapacitor, showed excellent performance in direct contact with physiological fluids such as artificial urine and phosphate buffered saline without any need of additional electrolytes, and exhibited almost no cytotoxicity during cycling tests in cell culture medium. Moreover, we demonstrated that the PEM-Dgel supercapacitor has greater charge-discharge cycling stability in physiological fluids than highly concentrated acid electrolyte solution which is normally used for supercapacitor operation. These conceptually new supercapacitors have the potential to be a platform technology for the creation of implantable energy storage devices for packageless applications directly utilizing biofluids.
The use of surface coating on biomaterials can render the original substratum with new functionalities that can improve the chemical, physical, and mechanical properties as well as enhance cellular cues such as attachment, proliferation, and differentiation. In this work, we combined biocompatible polydimethylsiloxane (PDMS) with a biomimetic polyelectrolyte multilayer (PEM) film made of poly(L-lysine) and hyaluronic acid (PLL/HA) for skeletal muscle tissue engineering. By microstructuring PDMS in grooves of a different width (5, 10, 30, and 100 μm) and by modulating the stiffness of the (PLL/HA) films, we guided skeletal muscle cell differentiation into myotubes. We found optimal conditions for both the formation of parallel-oriented myotubes and their maturation. Significantly, the myoblasts were collectively prealigned to the grooves before their differentiation. Before fusion, the highest aspect ratio and orientation of nuclei were observed for the 5 and 10 μm wide micropatterns. The formation of myotubes was observed regardless of the size of the micropatterns, and we found that their typical width was 10–12 μm. Their maturation was characterized by the immunolabeling of type II isomyosin. The amount of myosin striation was not affected by the topography, except for the 5 μm wide micropatterns. We highlighted the spatial constraints that led to an important nuclei deformation and further impairment of maturation within the 5 μm grooves. Altogether, our results show that the PEM film combined with PDMS is a powerful tool that is used for skeletal muscle engineering. This work opens perspectives for the development of skeletal muscle tissue in contact with films containing bioactive peptides or growth factors as well as for the study of pathogenic myotubes.
Paracetamol (acetaminophen, APAP) is a universally used analgesic and antipyretic agent. Considered safe at therapeutic doses, overdoses cause acute liver damage characterized by centrilobular hepatic necrosis. One of the major clinical problems of paracetamol-induced liver disease is the development of hemorrhagic alterations. Although hepatocytes represent the main target of the cytotoxic effect of paracetamol overdose, perturbations within the endothelium involving morphological changes of liver sinusoidal endothelial cells (LSECs) have also been described in paracetamol-induced liver disease. Recently, we have shown that paracetamol-induced liver damage is synergistically enhanced by the TRAIL signaling pathway. As LSECs are constantly exposed to activated immune cells expressing death ligands, including TRAIL, we investigated the effect of TRAIL on paracetamol-induced LSEC death. We here demonstrate for the first time that TRAIL strongly enhances paracetamol-mediated LSEC death with typical features of apoptosis. Inhibition of caspases using specific inhibitors resulted in a strong reduction of cell death. TRAIL appears to enhance paracetamol-induced LSEC death via the activation of the pro-apoptotic BH3-only proteins Bid and Bim, which initiate the mitochondrial apoptotic pathway. Taken together this study shows that the liver endothelial layer, mainly LSECs, represent a direct target of the cytotoxic effect of paracetamol and that activation of TRAIL receptor synergistically enhances paracetamol-induced LSEC death via the mitochondrial apoptotic pathway. TRAIL-mediated acceleration of paracetamol-induced cell death may thus contribute to the pathogenesis of paracetamol-induced liver damage.
liver sinusoidal endothelial cells (LSEC); paracetamol; TRAIL; Bcl-2 homologs; apoptosis
Background & Aims
After liver injury, bone marrow-derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPC and their effects on liver injury.
Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation).
Dimethylnitrosamine caused extensive denudation of LSEC at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSEC came from engrafted BM SPC. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSEC and reduced liver injury. Expression of hepatic VEGF mRNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSEC, and exacerbated dimethylnitrosamine injury.
BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.
endothelial progenitor cells; toxic hepatitis; animal model; liver damage
Tissue homeostasis and remodeling are processes that involve high turnover of biological macromolecules. Many of the waste molecules that are by-products or degradation intermediates of biological macromolecule turnover enter the circulation and are subsequently cleared by liver sinusoidal endothelial cells (LSEC). Besides the mannose receptor, stabilin-1 and stabilin-2 are the major scavenger receptors expressed by LSEC. To more clearly elucidate the functions of stabilin-1 and -2, we have generated mice lacking stabilin-1, stabilin-2, or both stabilin-1 and -2 (Stab1–/–Stab2–/– mice). Mice lacking either stabilin-1 or stabilin-2 were phenotypically normal; however, Stab1–/–Stab2–/– mice exhibited premature mortality and developed severe glomerular fibrosis, while the liver showed only mild perisinusoidal fibrosis without dysfunction. Upon kidney transplantation into WT mice, progression of glomerular fibrosis was halted, indicating the presence of profibrotic factors in the circulation of Stab1–/–Stab2–/– mice. While plasma levels of known profibrotic cytokines were unaltered, clearance of the TGF-β family member growth differentiation factor 15 (GDF-15) was markedly impaired in Stab1–/–Stab2–/– mice but not in either Stab1–/– or Stab2–/– mice, indicating that it is a common ligand of both stabilin-1 and stabilin-2. These data lead us to conclude that stabilin-1 and -2 together guarantee proper hepatic clearance of potentially noxious agents in the blood and maintain tissue homeostasis not only in the liver but also distant organs.
Adenovirus serotype 5 (Ad5) naturally infects the liver after intravenous injection, making it a candidate for hepatocyte-directed gene transfer. While Ad5 can be efficient, most of the dose is destroyed by liver Kupffer cells before it can reach hepatocytes. In contrast, Ad5 bearing the hexon from Ad6 (Ad5/6) evades Kupffer cells. While Ad5/6 dramatically increases hepatocyte transduction in BALB/c mice, it has surprisingly little effect on C57BL/6 mice. To determine the source of this strain-specific difference, the roles of Kupffer cells, liver sinusoidal endothelial cells (LSECs), hepatocytes, scavenger receptors, clotting factors, and immunoglobulins were analyzed. The numbers of Kupffer cells and LSECs, the level of clotting factor X, and hepatocyte infectibility did not differ between different strains of mice. In contrast, high levels of immunoglobulins correlated negatively with Ad5 liver transduction in different mouse strains. Removal of immunoglobulins by use of Rag-deficient mice restored Ad5 transduction to maximal levels. Removal of Kupffer cells by predosing or by testing in colony-stimulating factor knockout mice restored Ad5 transduction in the presence of immunoglobulins. Partial reconstitution of IgM in Rag mice resulted in significant reductions in liver transduction by Ad5 but not by Ad5/6. These data suggest a role for IgM-mediated clearance of Ad5 via Kupffer cells and may explain the mechanism by which Ad5/6 evades these cells. These mechanisms may play a vital role in Ad pharmacology in animals and in humans.
We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.
The integration of orthopedic implants with host bone presents a major challenge in joint arthroplasty, spinal fusion and tumor reconstruction. The cellular microenvironment can be programmed via implant surface functionalization allowing direct modulation of osteoblast adhesion, proliferation, and differentiation at the implant-bone interface. The development of layer-by-layer assembled polyelectrolyte multilayer (PEM) architectures has greatly expanded our ability to fabricate intricate nanometer to micron scale thin film coatings that conform to complex implant geometries. The in vivo therapeutic efficacy of thin PEM implant coatings for numerous biomedical applications has previously been reported. We have fabricated protamine-based PEM thin films that support the long-term proliferation and differentiation of pre-osteoblast cells on non-cross-linked film coated surfaces. These hydrophilic PEM functionalized surfaces with nanometer-scale roughness facilitated increased deposition of calcified matrix by osteoblasts in vitro, and thus offer the potential to enhance implant integration with host bone. The coatings can make an immediate impact in the osteogenic culture of stem cells and assessment of the osteogenic potential of new therapeutic factors.
The fungal pathogen Candida albicans can form biofilms on the surfaces of medical devices that are resistant to drug treatment and provide a reservoir for recurrent infections. The use of fungicidal or fungistatic materials to fabricate or coat the surfaces of medical devices has the potential to reduce or eliminate the incidence of biofilm-associated infections. Here, we report on (i) the fabrication of multilayered polyelectrolyte thin films (PEMs) that promote the surface-mediated release of an antifungal β-peptide and (ii) the ability of these films to inhibit the growth of C. albicans on film-coated surfaces. We incorporated a fluorescently labeled antifungal β-peptide into the structures of PEMs fabricated from poly-L-glutamic acid (PGA) and poly-L-lysine (PLL) using a layer-by-layer fabrication procedure. These films remained stable when incubated in culture media at 37 °C and released β-peptide gradually into solution for up to 400 hours. Surfaces coated with β-peptide-containing films inhibited the growth of C. albicans, resulting in a 20% reduction of cell viability after two hours and a 74% decrease in metabolic activity after seven hours when compared to cells incubated on PGA/PLL coated surfaces. In addition, β-peptide-containing films inhibited hyphal elongation by 55%. These results, when combined, demonstrate that it is possible to fabricate β-peptide-containing thin films that inhibit the growth and proliferation of C. albicans and provide the basis of an approach that could be used to inhibit the formation of C. albicans biofilms on film-coated surfaces. The layer-by-layer approach reported here could ultimately be used to coat the surfaces of catheters, surgical instruments, and other devices to inhibit drug-resistant C. albicans biofilm formation in clinical settings.
The liver may have a role in peripheral tolerance, by serving as a site for trapping, apoptosis and phagocytosis of activated T cells. It is not known which hepatic cells are involved in these processes. It was hypothesised that liver sinusoidal endothelial cells (LSEC) which are strategically placed for participation in the regulation of sinusoidal blood flow, and express markers involved in recognition, sequestration and apoptosis, may contribute to peripheral tolerance by inducing apoptosis of activated T cells.
By using immunoassays and western blot analysis, the fate of activated T cells when incubated with human LSEC isolated from normal healthy livers was investigated.
Evidence that activated (approximately 30%) but not non‐activated T cells undergo apoptosis on incubation with human LSEC in mixed cell cultures is provided. No difference in the results was observed when unstimulated and cytokine‐stimulated LSEC were used. T cell–LSEC contact is required for induction of apoptosis. Apoptosis induced by LSEC was associated with caspase 8 and 3 activity and strong expression of the proapoptotic molecule Bak. Transforming growth factor β (TGFβ) produced constitutively by LSEC is partly responsible for the caspase‐induced apoptosis, as neutralising antibodies to TGFβ markedly attenuated apoptosis, up regulated the antiapoptotic molecule Bcl‐2 and partially blocked caspase‐3 activity.
These findings broaden the potential role of LSEC in immune tolerance and homeostasis of the immune system. This study may provide insight for exploring the mechanisms of immune tolerance by liver allografts, immune escape by some liver pathogens including hepatitis C and pathogenesis of liver diseases.
Liver sinusoidal endothelial cells (LSECs) are specialized scavenger cells, with crucial roles in maintaining hepatic and systemic homeostasis. Under normal physiological conditions, the oxygen tension encountered in the hepatic sinusoids is in general considerably lower than the oxygen tension in the air; therefore, cultivation of freshly isolated LSECs under more physiologic conditions with regard to oxygen would expect to improve cell survival, structure and function. In this study LSECs were isolated from rats and cultured under either 5% (normoxic) or 20% (hyperoxic) oxygen tensions, and several morpho-functional features were compared.
Cultivation of LSECs under normoxia, as opposed to hyperoxia improved the survival of LSECs and scavenger receptor-mediated endocytic activity, reduced the production of the pro-inflammatory mediator, interleukin-6 and increased the production of the anti-inflammatory cytokine, interleukin-10. On the other hand, fenestration, a characteristic feature of LSECs disappeared gradually at the same rate regardless of the oxygen tension. Expression of the cell-adhesion molecule, ICAM-1 at the cell surface was slightly more elevated in cells maintained at hyperoxia. Under normoxia, endogenous generation of hydrogen peroxide was drastically reduced whereas the production of nitric oxide was unaltered. Culture decline in high oxygen-treated cultures was abrogated by administration of catalase, indicating that the toxic effects observed in high oxygen environments is largely caused by endogenous production of hydrogen peroxide.
Viability, structure and many of the essential functional characteristics of isolated LSECs are clearly better preserved when the cultures are maintained under more physiologic oxygen levels. Endogenous production of hydrogen peroxide is to a large extent responsible for the toxic effects observed in high oxygen environments.