Murine Liver Sinusoidal Endothelial Cells Research Articles

Mechanotransduction-induced glycolysis epigenetically regulates a CXCL1-dominant angiocrine signaling program in liver sinusoidal endothelial cells invitro and invivo.

Liver sinusoidal endothelial cells (LSECs) are ideally situated to sense stiffness and generate angiocrine programs that potentially regulate liver fibrosis and portal hypertension. We explored how specific focal adhesion (FA) proteins parlay LSEC mechanotransduction into stiffness-induced angiocrine signaling invitro and invivo. Primary human and murine LSECs were placed on gels with incremental stiffness (0.2 kPa vs. 32 kPa). Cell response was studied by FA isolation, actin polymerization assay, RNA-sequencing and electron microscopy. Glycolysis was assessed using radioactive tracers. Epigenetic regulation of stiffness-induced genes was analyzed by chromatin-immunoprecipitation (ChIP) analysis of histone activation marks, ChIP sequencing and circularized chromosome conformation capture (4C). Mice with LSEC-selective deletion of glycolytic enzymes (Hk2fl/fl/Cdh5cre-ERT2) or treatment with the glycolysis inhibitor 3PO were studied in portal hypertension (partial ligation of the inferior vena cava, pIVCL) and early liver fibrosis (CCl4) models. Glycolytic enzymes, particularly phosphofructokinase 1 isoform P (PFKP), are enriched in isolated FAs from LSECs on gels with incremental stiffness. Stiffness resulted in PFKP recruitment to FAs, which paralleled an increase in glycolysis. Glycolysis was associated with expansion of actin dynamics and was attenuated by inhibition of integrin β1. Inhibition of glycolysis attenuated a stiffness-induced CXCL1-dominant angiocrine program. Mechanistically, glycolysis promoted CXCL1 expression through nuclear pore changes and increases in NF-kB translocation. Biochemically, this CXCL1 expression was mediated through spatial re-organization of nuclear chromatin resulting in formation of super-enhancers, histone acetylation and NF-kB interaction with the CXCL1 promoter. Hk2fl/fl/Cdh5cre-ERT2 mice showed attenuated neutrophil infiltration and portal hypertension after pIVCL. 3PO treatment attenuated liver fibrosis in a CCl4 model. Glycolytic enzymes are involved in stiffness-induced angiocrine signaling in LSECs and represent druggable targets in early liver disease. Treatment options for liver fibrosis and portal hypertension still represent an unmet need. Herein, we uncovered a novel role for glycolytic enzymes in promoting stiffness-induced angiocrine signaling, which resulted in inflammation, fibrosis and portal hypertension. This work has revealed new targets that could be used in the prevention and treatment of liver fibrosis and portal hypertension.

Tuning of Liver Sieve: The Interplay between Actin and Myosin Regulatory Light Chain Regulates Fenestration Size and Number in Murine Liver Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs) facilitate the efficient transport of macromolecules and solutes between the blood and hepatocytes. The efficiency of this transport is realized via transcellular nanopores, called fenestrations. The mean fenestration size is 140 ± 20 nm, with the range from 50 nm to 350 nm being mostly below the limits of diffraction of visible light. The cellular mechanisms controlling fenestrations are still poorly understood. In this study, we tested a hypothesis that both Rho kinase (ROCK) and myosin light chain (MLC) kinase (MLCK)-dependent phosphorylation of MLC regulates fenestrations. We verified the hypothesis using a combination of several molecular inhibitors and by applying two high-resolution microscopy modalities: structured illumination microscopy (SIM) and scanning electron microscopy (SEM). We demonstrated precise, dose-dependent, and reversible regulation of the mean fenestration diameter within a wide range from 120 nm to 220 nm and the fine-tuning of the porosity in a range from ~0% up to 12% using the ROCK pathway. Moreover, our findings indicate that MLCK is involved in the formation of new fenestrations—after inhibiting MLCK, closed fenestrations cannot be reopened with other agents. We, therefore, conclude that the Rho-ROCK pathway is responsible for the control of the fenestration diameter, while the inhibition of MLCK prevents the formation of new fenestrations.

AFM image analysis of porous structures by means of neural networks

In this work, we presented a method of finding and characterising transmembrane porous structures, called fenestrations, with the help of convolutional neural networks. Case studies are performed on high resolution AFM images of murine liver sinusoidal endothelial cells (LSECs). At first, we evaluated different kinds of noise occurring in the LSEC AFM measurements. Next, we proposed a schematic structure of the neural network suitable for our purpose. We examined different loss functions, optimising the accuracy of fenestration detection. Finally, we presented the method of rough calculation of fenestration size distributions.We demonstrated that the accuracy of this method surpasses 90%. Furthermore, it is fast, not sensitive to the chosen image contrast and fully deterministic. The simple scheme can be easily modified to different objects of interest, which promotes the use of neural networks as a universal tool for the analysis of various kinds of microscopy images.

Sensing of Liver Iron Content Requires Cell-Cell Communication between Hepatocytes and Liver Sinusoidal Endothelial Cells

The liver stores iron and senses systemic and tissue iron availability. Hepatocytes control iron homeostasis by producing the peptide hormone hepcidin that controls dietary iron absorption and iron release from intracellular stores. Recent data challenged the exclusive role of hepatocytes in controlling iron levels. Indeed, liver sinusoidal endothelial cells (LSECs) increase BMP2 and BMP6 levels in response to iron, which control hepcidin expression in a paracrine manner. However the molecular mechanism(s) of how BMPs respond to iron levels remain unknown. We established primary murine LSEC cultures and exposed these to iron sources. Unexpectedly, BMP2 mRNA expression is strongly reduced by iron treatment, while BMP6 levels are only mildly increased. This finding suggests that intracellular iron content cannot directly activate BMP2 transcription and only slightly contribute to BMP6 upregulation in LSEC cultures. However, if LSECs are co-cultured with iron-loaded primary hepatocytes the expression of BMP2 and BMP6 is increased and the fold induction of BMP6 is greater compared to LSECs cultured alone, suggesting that the iron status of hepatocytes instructs the LSEC BMP response. These data are supported by findings in a genetic mouse model of iron overload (Slc40a1C326S/C326S). Hepatocytes isolated from Slc40a1C326S/C326S mice display an iron-loaded molecular signature and the expected low mRNA expression of Transferrin Receptor 1 (Tfr1). By contrast, LSECs show high expression of Tfr1, indicating intracellular iron deficiency. Despite this, hepatic BMP levels are increased, suggesting that BMP2 and BMP6 expression are directly related to the intracellular iron content of hepatocytes but not LSECs. RNA-sequencing of isolated hepatic cell populations is ongoing to identify putative hepatocyte regulators involved in the iron-mediated BMP2 and BMP6 regulation. Disclosures Muckenthaler: Silence Therapeutics: Consultancy; Novartis: Research Funding.

Application of a layered model for determination of the elasticity of biological systems

Elasticity of biological systems is considered to be an important property that might be related to functional or pathological changes. Therefore, careful study and detailed understanding of cell and tissue elasticity is crucial for correct description of their functioning. Atomic Force Microscopy (AFM) is a powerful technique, which allows for determination of the physical properties, such as elasticity, of soft-matter systems in nano-scale. An important step in AFM elasticity studies is a proper interpretation of experimental data. Two most frequently used theoretical schemes applied to determine elasticity are due to Hertz and Sneddon, which are effectively one-parameter models. In this work, we go beyond this approach. Firstly, as elasticity is a local property, we extract from the slope of experimental force-indentation curve an elasticity parameter, which varies with indentation depth. Then secondly, we find best approximation of this parameter by applying the two-layer model with four effective parameters, as proposed by Kovalev. This method is employed to the experimental data taken on murine liver sinusoidal endothelial cells in non-alcoholic fatty liver disease model. The obtained results show additional effects, not seen within the traditional, simplified scheme. Namely, the elasticity of the first layer does not change its value in the model of non-alcoholic fatty liver disease, but the increase of stiffness is noticed in second layer. The second goal of this article is to reveal and discuss the differences between traditional approaches and the one being presented. The deviations from the original assumptions are analysed and the corresponding restrictions on utility of theoretical models are presented.

Treprostinil reduces endothelial damage in murine sinusoidal obstruction syndrome

Sinusoidal obstruction syndrome (SOS) is a major complication after hematopoietic stem cell transplantation and belongs to a group of diseases increasingly identified as transplant-related systemic endothelial disease. Administration of defibrotide affords some protection against SOS, but the effect is modest. Hence, there is unmet medical need justifying the preclinical search for alternative approaches. Prostaglandins exert protective actions on endothelial cells of various vascular beds. Here, we explored the therapeutic potential of the prostacyclin analog treprostinil to prevent SOS. Treprostinil acts via stimulation of IP, EP2, and EP4 receptors, which we detected in murine liver sinusoidal endothelial cells (LSECs). Busulfan-induced cell death was reduced when pretreated with treprostinil in vitro. In a murine in vivo model of SOS, concomitantly administered treprostinil caused lower liver weight-to-body weight ratios indicating liver protection. Histopathological changes were scored to assess damage to liver sinusoidal endothelial cells, to hepatocytes, and to the incipient fibrotic reaction. Treprostinil indeed reduced sinusoidal endothelial cell injury, but this did not translate into reduced liver cell necrosis or fibrosis. In summary, our observations provide evidence for a beneficial effect of treprostinil on damage to LSECs but unexpectedly treprostinil was revealed as a double-edged sword in SOS.Key messagesMurine liver sinusoidal endothelial cells (LSECs) express prostanoid receptors.Treprostinil reduces busulfan-induced cell death in vitro.Treprostinil lowers liver weight-to-body weight ratios in mice.Treprostinil positively affects LSECs in mice but not hepatic necrosis/fibrosis.

Very small superparamagnetic iron oxide nanoparticles: Long-term fate and metabolic processing in atherosclerotic mice

We investigated the biotransformation of very small superparamagnetic iron oxide nanoparticles (VSOP) in atherosclerotic LDLR−/− mice. Transmission electron microscopy revealed an uptake of VSOP not only by macrophages but also by endothelial cells in liver, spleen, and atherosclerotic lesions and their accumulation in the lysosomal compartment. Using magnetic particle spectroscopy (MPS), we show that the majority of VSOP's superparamagnetic iron was degraded within 28 days. MPS spectrum shape indicated changes in the magnetic properties of VSOP during the biodegradation process. Experiments with primary murine bone marrow derived macrophages, primary murine liver sinusoidal endothelial cells, and primary human aortic endothelial cells demonstrated that loading with VSOP induced a differential response of cellular iron homeostasis mechanisms with increased levels of ferritin and iron transport proteins in macrophages and increased levels of ferritin in endothelial cells.

Atomic Force Microscopy Reveals the Dynamic Morphology of Fenestrations in Live Liver Sinusoidal Endothelial Cells

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Quantification of fenestrations in liver sinusoidal endothelial cells by atomic force microscopy

Liver sinusoidal endothelial cells present unique morphology characterized by the presence of transmembrane pores called fenestrations. The size and number of fenestrations in live cells change dynamically in response to variety of chemical and physical factors. Although scanning electron microscopy is a well-established method for investigation of fixed liver sinusoidal endothelial cells morphology, atomic force microscopy is the interesting alternative providing detailed 3D topographical information. Moreover, simple sample preparation, only by wet-fixation, minimizing sample preparation artifacts enable high-resolution atomic force microscopy-based measurements. In this work, we apply imaging methods based on atomic force microscopy, to describe characteristic features of glutaraldehyde-fixed primary murine liver sinusoidal endothelial cells, namely: mean fenestration diameter, porosity, and fenestrations frequency. We also investigate the effect of different tip apex radius on evaluation of single fenestration diameter.By quantitative description of fenestrations, we demonstrate that atomic force microscopy became a well competing tool for nondestructive quantitative investigation of the liver sinusoidal endothelial cell morphology.

Methods for Isolation and Purification of Murine Liver Sinusoidal Endothelial Cells: A Systematic Review.

To study the biological functions of liver sinusoidal endothelial cells (LSEC) and to identify their interplay with blood or liver cells, techniques allowing for the isolation and purification of LSEC have been developed over the last decades. The objective of the present review is to summarize and to compare the efficiency of existing methods for isolating murine LSEC. Toward this end, the MEDLINE database was searched for all original articles describing LSEC isolation from rat and mouse livers. Out of the 489 publications identified, 23 reported the main steps and outcomes of the procedure and were included in our review. Here, we report and analyse the technical details of the essential steps of the techniques used for LSEC isolation. The correlations between the prevalence of some steps and the efficiency of LSEC isolation were also identified. We found that centrifugal elutriation, selective adherence and, more recently, magnetic-activated cell sorting were used for LSEC purification. Centrifugal elutriation procured high yields of pure LSEC (for rats 30–141.9 million cells for 85–98% purities; for mice 9–9.25 million cells for >95% purities), but the use of this method remained limited due to its high technical requirements. Selective adherence showed inconsistent results in terms of cell yields and purities in rats (5–100 million cells for 73.7–95% purities). In contrast, magnetic-activated cell sorting allowed for the isolation of highly pure LSEC, but overall lower cell yields were reported (for rats 10.7 million cells with 97.6% purity; for mice 0.5–9 million cells with 90–98% purities). Notably, the controversies regarding the accuracy of several phenotypic markers for LSEC should be considered and their use for both magnetic sorting and characterization remain doubtful. It appears that more effort is needed to refine and standardize the procedure for LSEC isolation, with a focus on the identification of specific antigens. Such a procedure is required to identify the molecular mechanisms regulating the function of LSEC and to improve our understanding of their role in complex cellular processes in the liver.

Standardized isolation and culture of murine liver sinusoidal endothelial cells.

Isolation of murine liver sinusoidal endothelial cells (LSECs) is an exacting and finicky procedure. After exhaustive standardization, we were able to devise an easily reproducible protocol which produced consistent results. Moreover, we scripted a protocol which clarifies even the smallest of steps, following which isolation of LSECs is made significantly easier. Using the standardized LSEC isolation protocol herein, we demonstrated that the bacterial toxin pyocyanin (from Pseudomonas aeruginosa) induced a significant dose-dependent reduction in LSEC porosity, this being preventable by the enzyme catalase, but not by the enzyme superoxide dismutase.

Adoptive Transfer of Allogeneic Liver Sinusoidal Endothelial Cells Specifically Inhibits T-Cell Responses to Cognate Stimuli

Although it is well known that liver allografts are often accepted by recipients, leading to donor-specific tolerance of further organ transplants, the underlying mechanisms remain unclear. We had previously used an in vitro model and showed that mouse liver sinusoidal endothelial cells (LSECs) selectively suppress allospecific T-cells across major histocompatibility complex (MHC) barriers. In the present study, we established an in vivo model for evaluating the immunomodulatory effects of allogeneic LSECs on corresponding T-cells. Allogeneic BALB/cA LSECs were injected intraportally into recombination activating gene 2 γ-chain double-knockout (RAG2/gc-KO, H-2(b)) mice lacking T, B, and natural killer (NK) cells. In order to facilitate LSEC engraftment, the RAG2/gc-KO mice were injected intraperitoneally with monocrotaline 2 days before the adoptive transfer of LSECs; this impaired the host LSECs, conferring a proliferative advantage to the transplanted LSECs. After orthotopic allogeneic LSEC engraftment, the RAG2/gc-KO mice were immune reconstituted intravenously with C57BL/6 splenocytes. After immune reconstitution, mixed lymphocyte reaction (MLR) assay using splenocytes from the recipients revealed that specific inhibition of host CD4(+) and CD8(+) T-cell proliferation was greater in response to allostimulation with irradiated BALB/cA splenocytes rather than to stimulation with irradiated third party SJL/jorllco splenocytes. This inhibitory effect was attenuated by administering anti-programmed death ligand 1 (PD-L1) monoclonal antibody during immune reconstitution in the above-mentioned mice, but not in RAG2/gc-KO mice engrafted with Fas ligand (FasL)-deficient BALB/cA LSECs. Furthermore, engraftment of allogeneic BALB/cA LSECs significantly prolonged the survival of subsequently grafted cognate allogeneic BALB/cA hearts in RAG2/gc-KO mice immune reconstituted with bone marrow transplantation from C57BL/6 mice. In conclusion, murine LSECs have been proven capable of suppressing T-cells with cognate specificity for LSECs in an in vivo model. The programmed death 1/PD-L1 pathway is likely involved in these suppressive effects.

Inhibition of inflammatory CD4 T cell activity by murine liver sinusoidal endothelial cells

The liver can mitigate the inflammatory activity of infiltrating T cells by mechanisms that are not entirely clear. Here we investigated the role of liver sinusoidal endothelial cells (LSECs) in regulating the activity of inflammatory CD4 T cells. Interactions between T helper (Th) 1 or Th17 cells and LSEC were studied by intravital microscopy and by in vitro stimulation assays. Circulating CD4 T cells established lasting and repeated interactions with liver endothelium in vivo. Stimulation of Th1 and Th17 cells by LSEC greatly inhibited their capacity to secrete interferon-γ or interleukin-17 in vitro; in contrast, stimulation by dendritic cells (DCs) resulted in considerable secretion of both cytokines. Cytokine release by Th1 or Th17 cells seemed to be actively suppressed by LSEC, as indicated by the inhibition of cytokine secretion even in the presence of Th1- and Th17-promoting DC. This inhibition of CD4 T cell effector function seemed to depend on the dominance of inhibitory over activating co-stimulatory signals on LSEC, since (1) cytokine secretion could be restored by increased CD28 co-activation; (2) LSEC from interleukin-10(-/-) mice, which manifest increased activating signals, such as MHC II, and decreased inhibitory signals, such as PD-L1, failed to suppress cytokine secretion; and (3) cytokine secretion by Th1 or Th17 cells that lacked PD-1, the ligand for inhibitory PD-L1, could not be suppressed by LSEC. LSEC inhibit inflammatory cytokine secretion of Th1 and Th17 effector CD4 T cells in dependence of interleukin-10 and PD-1.

Liver Sinusoidal Endothelial Cells Promote B Lymphopoiesis From Primitive Hematopoietic Cells

Although the bone marrow (BM) microenvironment is the main inducer niche of early B lymphopoiesis during the adult life, other extramedullar microenvironments, such as the liver, may also have potential for supporting B-cell development. Previously, we reported that murine liver sinusoidal endothelial cells (LSECs) support in vitro and in vivo hematopoietic stem cell (HSC) proliferation and myeloid differentiation. In the present study, we investigated the capacity of LSEC to promote B lymphopoiesis from BM progenitor lineage-negative (Lin(-)) cells. Murine BM Lin(-) cells were co-cultured with LSEC, in the absence of exogenous cytokines. B cells were characterized by flow cytometry and cytokine expression by RT-PCR. We show that BM Lin(-) cells differentiated to early B-lymphoid progenitors (B220(+)) and subsequently to mature (CD19(+)) B cells. Functional studies showed the presence of a high number of non-adherent cells (NACs), collected from lipopolysaccharide (LPS)-treated Lin(-)/LSEC co-cultures, expressing IgM on their surface (sIgM). Colony formation from NAC was observed in the presence of IL-7 (CFU-IL-7). LSEC constitutively express IL-7, Flt-3L, and SCF at the mRNA level, and VCAM-1 on their surface, which may explain the capacity of these cells to promote B lymphopoiesis. These data demonstrate that LSEC promote all stages of B lymphopoiesis. To our knowledge, this is the first report that LSEC constitute an in vitro microenvironment for B lymphopoiesis. Further studies will establish whether LSEC can serve in vivo as a B-lymphopoietic niche under physiological or pathological condition, or when HSC are mobilized.

Virally Infected Mouse Liver Endothelial Cells Trigger CD8 + T-Cell Immunity

Dendritic cell activation through ligation of pattern recognition receptors leading to full functional maturation causes induction of CD8(+) T-cell immunity through increased delivery of costimulatory signals instead of tolerance. Here we investigate whether organ-resident antigen-presenting cells, such as liver sinusoidal endothelial cells (LSECs), also switch from tolerogenic to immunogenic CD8(+) T-cell activation upon such stimulation. Murine LSECs were isolated by immunomagnetic separation and analyzed for functional maturation upon triggering pattern recognition receptors or viral infection employing gene expression analysis and T cell coculture assays. In vivo relevance of the findings was confirmed with bone-marrow chimeric animals. LSECs expressed numerous pattern recognition receptors that allowed for sentinel function, but ligand-induced activation of these receptors was not sufficient to overcome tolerance induction of CD8(+) T cells. Importantly, viral infection with murine cytomegalovirus caused functional maturation of antigen-presenting LSECs and was sufficient to promote antigen-specific differentiation into effector CD8(+) T cells in the absence of dendritic cells and independent of CD80/86. These results shed new light on the generation of organ-specific immunity and may contribute to overcoming tolerance in relevant situations, such as cancer.

Systemic antigen cross-presented by liver sinusoidal endothelial cells induces liver-specific CD8 T-cell retention and tolerization

Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance. Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.

The mannose receptor on murine liver sinusoidal endothelial cells is the main denatured collagen clearance receptor

The purpose of this study was to identify the receptor responsible for endocytosis of denatured collagen from blood. The major site of clearance of this material (at least 0.5 g/day in humans) is a receptor on liver sinusoidal endothelial cells (LSECs). We have now identified an 180-kDa endocytic receptor on LSECs, peptide mass fingerprinting of which revealed it to be the mannose receptor. Challenge of mannose-receptor knockout mice and their cultured LSECs revealed significantly reduced blood clearance and a complete absence of LSEC endocytosis of denatured collagen. Organ analysis of wild-type versus knockout mice after injection of denatured collagen revealed significantly reduced liver uptake in the knockout mice. Clearance/endocytosis of ligands for other receptors in these animals was as that for wild-type mice, and denatured collagen uptake in wild-type mice was not affected by other ligands of the mannose receptor, namely mannose and mannan. Furthermore, unlike that of mannose and mannan, endocytosis of denatured collagen by the mannose receptor is calcium independent. This suggests that the binding site for denatured collagen is distinct from that for mannose/mannan. Mannose receptors on LSECs appear to have less affinity for circulating triple helical type I collagen. The mannose receptor is the main candidate for being the endocytic denatured collagen receptor on LSECs.

Crosstalk between Murine Liver Sinusoidal Endothelial Cells (LSEC) and Hematopoietic Stem Cells during In Vitro Hematopoiesis.

Crosstalk between murine liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells during in vitro hematopoiesis. G. Luna, J. E. Cardier. Laboratorio de Patologia Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas (IVIC). Apartado 21827, Caracas, VenezuelaWe have showed that at the liver there is a specific hematopoietic microenvironment, constituted by the liver sinusoid endothelial cells (LSEC), which are capable to support, in vitro and in vivo, not only the proliferation but also the differentiation and survival of hematopoietic stem cells (HSC). The ability of LSEC to support hematopoiesis could be related to specific hematopoietic molecules expressed by these cells. In this study, we investigate the expression of some cell adhesion molecules (VCAM-1, ICAM-1, and the integrin a4b1 ), and early acting hematopoietic cytokines (IL-6 and GM-CSF), on LSEC cocultured with HSC. The expression of VCAM-1, ICAM-1, α4β1, IL6 and GM-SCF was increased on LSEC cocultured with HSC. In contrast, a significant decrease in the expression of IL-6 and GM-CSF in the HSC derived from the same cocultures was observed. The blockade of VCAM-1 on LSEC reduced significantly the adhesion of HSC to LSEC monolayers, suggesting that this molecule is involved in the binding of HSC to LSEC microenvironments. There were not changes in the expression of the molecules evaluated when the LSEC and HSC were co-cultured in non-contact conditions, suggesting that soluble factors do not participate in regulating the expression of these molecules. Our data shows that during in vitro hematopoiesis, LSEC are activated to express molecules associated with the hematopoiesis process. LSEC activation is regulated by the contact between these cells and HSC. By expressing critical hematopoietic microenvironment molecules, LSEC may regulate the proliferation and differentiation of HSC, during liver extramedullary hematopoiesis.

Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium: A Role for Capturing Hepatitis C Virus Particles

Expression of DC-SIGN and DC-SIGNR on Human Sinusoidal Endothelium: A Role for Capturing Hepatitis C Virus Particles

High Efficiency and Long-Term Foreign Gene Expression in Cultured Liver Sinusoidal Endothelial Cells by Retroviral Transduction

The liver sinusoidal endothelial cells (LSECs) constitute a very specialized endothelium. Due to their multiple functions and privileged location in the liver, these cells constitute an excellent target for gene therapy. In this work, the authors investigate the efficiency of retroviral gene transduction as a method for in vitro gene delivery into murine LSECs. Gene transduction into murine LSECs was performed using the PCMMP-eGFP/pIK-MLVgp retrovirus pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-g), containing eGFP as a reporter gene. Retroviral transduction resulted in a high efficiency of gene transfer (99%) and stable expression of eGFP in LSECs. The retroviral transduction protocol did not affect the morphology or expression of endothelial cell markers or the biological functions of LSECs. The authors have developed conditions for high-efficiency and stable retroviral gene transduction of LSECs. These results raise the possibility of liver gene therapy using LSECs as vehicle for the delivery of therapeutic proteins by means of retroviral vectors.