Receptor-Mediated Endocytosis of VEGF-A in Rat Liver Sinusoidal Endothelial Cells.

BACKGROUND AND AIMS: Vascular endothelial growth factor (VEGF) receptors (VEGFR1 and VEGFR2) bind VEGF-A with high affinity. This study sought to determine the relative contributions of these two receptors to receptor-mediated endocytosis of VEGF-A and to clarify their endocytic itineraries in rat liver sinusoidal endothelial cells (LSECs). METHODS: Isolated LSECs and radiolabeled VEGF-A were used to examine surface binding and receptor-mediated endocytosis. Quantitative real time RT-PCR (Q-RT-PCR) and Western blotting were applied to demonstrate receptor expression. RESULTS: Q-RT-PCR analysis showed that VEGFR1 and VEGFR2 mRNA were expressed in LSECs. Ligand saturation analysis at 4°C indicated two different classes of [125I]-VEGFA binding sites on LSECs with apparent dissociation constants of 8 and 210 pM. At 37°C, LSECs efficiently took up and degraded [125I]-VEGF-A for at least 2 hours. Uptake of [125I]-VEGF-A by LSECs was blocked by dynasore that inhibits dynamin-dependent internalization, whereas inhibition of cysteine proteases by leupeptin inhibited degradation without affecting the uptake of [125I]-VEGF-A, suggesting that it is degraded following transport to lysosomes. Incubation of LSECs in the continued presence of a saturating concentration of unlabeled VEGF-A at 37°C was associated with a loss of as much as 75% of the total VEGFR2 within 30 min as shown by Western blot analysis, whereas there was no appreciable decrease in protein levels for VEGFR1 after 120 min incubation, suggesting that VEGF-A stimulation downregulates VEGFR2, but not VEGFR1, in LSECs. This possibility was supported by the observation that a hexapeptide that specifically blocks VEGF-A binding to VEGFR1 caused a marked reduction in the uptake of [125I]-VEGF-A, whereas a control peptide had no effect. Finally, live cell imaging studies using a fluorescently labeled anti-VEGFR2 antibody showed that VEGFR2 was transported via early and late endosomes to reach endolysosomes where degradation of the VEGFR2 takes place. CONCLUSION: Our studies suggest that, subsequent to VEGF-A binding and internalization, the unoccupied VEGFR1 may recycle to the cell surface allowing its reutilization, whereas the majority of the internalized VEGFR2 is targeted for degradation.

Enhanced activity of lysosomal proteases in activated rat hepatic stellate cells is associated with a concomitant increase in the number of the mannose-6-phosphate/insulin-like growth factor II receptor.

Activated hepatic stellate cells (HSCs) play a central role during hepatic tissue repair through their influence on extracellular matrix remodelling. We have determined whether the activity levels of cathepsin B and D are affected by in vitro activation of rat HSCs, and whether the enzymes were released from the cells. Furthermore, given the important role of the mannose-6-phosphate/insulin-like growth factor II receptor (M6P/IGF-IIR) in the intracellular transport of lysosomal enzymes, we have examined whether changes in the activity of these proteases were associated with parallel changes in the level of the M6P/IGF-IIR. The activity of cathepsin B and D increased ∼4 times between 2 and 8 days of HSC culture. This result was supported by analysing mRNA expression by RT-PCR. The cells released the enzymes into the culture medium, amounting to ∼10% of the cell-associated activity over 24 h. The release of enzymes was not affected by reducing medium pH from 7.4 to 6.2, indicating that the enzymes were transported to the medium independently of the M6P/IGF-II-R. The released cathepsin B was mostly in the inactive proenzyme form. HSC activation led to a particularly large increase in M6P/IGF-IIR expression. A large proportion of the receptors was located on the cell surface and was found to be very suitable for measuring endocytosis of (125) I-IGF-II. The results show that the endocytic activity increased in parallel with the increase in surface receptors and activity of lysosomal enzymes. Degradation of the ligand was reduced by inhibitors of lysosomal proteases and therefore took place in lysosomes.

The scavenger endothelial cell: a new player in homeostasis and immunity.

To maintain homeostasis, the animal body is equipped with a powerful system to remove circulating waste. This review presents evidence that the scavenger endothelial cell (SEC) is responsible for the clearance of blood-borne waste macromolecules in vertebrates. SECs express pattern-recognition endocytosis receptors (mannose and scavenger receptors), and in mammals, the endocytic Fc gamma-receptor IIb2. This cell type has an endocytic machinery capable of super-efficient uptake and degradation of physiological and foreign waste material, including all major classes of biological macromolecules. In terrestrial vertebrates, most SECs line the wall of the liver sinusoid. In phylogenetically older vertebrates, SECs reside instead in heart, kidney, or gills. SECs, thus, by virtue of their efficient nonphagocytic elimination of physiological and microbial substances, play a critical role in the innate immunity of vertebrates. In major invertebrate phyla, including insects, the same function is carried out by nephrocytes. The concept of a dual-cell principle of waste clearance is introduced to emphasize that professional phagocytes (macrophages in vertebrates; hemocytes in invertebrates) eliminate larger particles (>0.5 µm) by phagocytosis, whereas soluble macromolecules and smaller particles are eliminated efficiently and preferentially by clathrin-mediated endocytosis in nonphagocytic SECs in vertebrates or nephrocytes in invertebrates. Including these cells as important players in immunology and physiology provides an additional basis for understanding host defense and tissue homeostasis.

Affinity and kinetics of proprotein convertase subtilisin/kexin type 9 binding to low-density lipoprotein receptors on HepG2 cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that regulates the number of cell surface low-density lipoprotein receptors (LDLRs) and the levels of low-density lipoprotein cholesterol in plasma. Intact cells have not previously been used to determine the characteristics of binding of PCSK9 to LDLR. Using PCSK9 iodinated by the tyramine cellobiose (TC) method ([(125)I]TC-PCSK9), we measured the affinity and kinetics of binding of PCSK9 to LDLR on HepG2 cells at 4 °C. The extent of [(125)I]TC-PCSK9 binding increased as cell surface LDLR density increased. Unlabeled wild-type and two gain-of-function mutants of PCSK9 reduced binding of [(125)I]TC-PCSK9. The Scatchard plot of the binding-inhibition curve was curvilinear, indicative of high-affinity and low-affinity sites for PCSK9 binding on HepG2 cells. Nonlinear regression analysis of the binding data also indicated that a two-site model better fitted the data. The time course of [(125)I]TC-PCSK9 binding showed two phases in the association kinetics. Dissociation of [(125)I]TC-PCSK9 also occurred in two phases. Unlabeled PCSK9 accelerated the dissociation of [(125)I]TC-PCSK9. At low pH, only one phase of dissociation was apparent. Furthermore, the dissociation of [(125)I]TC-PCSK9 under pre-equilibrium conditions was faster than under equilibrium conditions. Overall, the data suggest that PCSK9 binding to cell surface LDLR cannot be described by a simple bimolecular reaction. Possible interpretations that can account for these observations are discussed.

Deficiency of activating Fcγ-receptors reduces hepatic clearance and deposition of IC and increases CIC levels in mercury-induced autoimmunity.

BACKGROUND: Inorganic mercury (Hg) induces a T-cell dependent, systemic autoimmune condition (HgIA) where activating Fcγ-receptors (FcγRs) are important for the induction. In this study we examined the influence of activating FcγRs on circulating levels and organ localization of immune complexes (IC) in HgIA. METHODS AND PRINCIPAL FINDINGS: Mercury treated BALB/c wt mice showed a significant but modest increase of circulating IC (CIC) from day 12 until day 18 and day 35 for IgG2a- and IgG1- CIC, respectively. Mercury-treated mice lacking the trans-membrane γ-chain of activating FcγRs (FcRγ(-/-)) had significantly higher CIC levels of both IgG1-CIC and IgG2a-CIC than wt mice during the treatment course. The hepatic uptake of preformed CIC was significantly more efficient in wt mice compared to FcγR(-/-) mice, but also development of extrahepatic tissue IC deposits was delayed in FcRγ(-/-) mice. After 35 days of Hg treatment the proportion of immune deposits, as well as the amounts was significantly reduced in vessel FcRγ(-/-) mice compared to wt mice. CONCLUSIONS: We conclude that mice lacking functional activating FcγRs respond to Hg with increased levels and altered quality of CIC compared with wt mice. Lack of functional activating FcγRs delayed the elimination of CIC, but also significantly reduced extrahepatic tissue localization of CIC.

Up-regulation of uPARAP/Endo180 during culture activation of rat hepatic stellate cells and its presence in hepatic stellate cell lines from different species.

BACKGROUND: The urokinase plasminogen activator receptor associated protein (uPARAP)/Endo180 is a novel endocytic receptor that mediates collagen uptake and is implicated to play a role in physiological and pathological tissue-remodelling processes by mediating intracellular collagen degradation. RESULT: This study investigates the expression of uPARAP/Endo180 protein and messenger RNA in primary rat hepatic stellate cell (HSC) cultures. The results show that uPARAP/Endo180 protein is not expressed in freshly isolated HSCs or during the first few days of culture while the cells still display quiescent features. In contrast, uPARAP/Endo180 protein is expressed early during HSC activation when cells are transdifferentiated into myofibroblast-like cells. Very low levels of uPARAP/Endo180 mRNA are detectable during the first days of culture but uPARAP/Endo180 mRNA is strongly up-regulated with increasing time in culture. Moreover, endocytic uptake of denatured collagen increases as transdifferentiation proceeds over time and correlates with increased expression of uPARAP/Endo180. Finally, analysis of uPARAP/Endo180 expression in four hepatic stellate cell lines from three different species showed that all these cell lines express uPARAP/Endo180 and are able to take up denatured collagen efficiently. CONCLUSION: These results demonstrate that uPARAP/Endo180 expression by rat HSCs is strongly up-regulated during culture activation and identify this receptor as a feature common to culture-activated HSCs.

Receptor-mediated endocytosis of immune complexes in rat liver sinusoidal endothelial cells is mediated by FcgammaRIIb2.

UNLABELLED: Liver sinusoidal endothelial cells (LSECs) display a number of receptors for efficient uptake of potentially injurious molecules. The receptors for the Fc portion of immunoglobulin G (IgG) antibodies (FcgammaRs) regulate a number of physiological and pathophysiological events. We used reverse transcription polymerase chain reaction (RT-PCR) and Western blotting to determine the expression of different types of FcgammaRs in LSECs. Biochemical approaches and immunofluorescence microscopy were used to characterize the FcgammaR-mediated endocytosis of immune complexes (ICs). FcgammaRIIb2 was identified as the main receptor for the efficient uptake of ICs in LSECs. The receptor was shown to use the clathrin pathway for IC uptake; however, the association with lipid rafts may slow the rate of its internalization. Moreover, despite trafficking through lysosomal integral membrane protein-II (LIMP-II)-containing compartments, the receptor was not degraded. Finally, it was shown that the receptor recycles to the cell surface both with and without IC. CONCLUSION: FcgammaRIIb2 is the main receptor for endocytosis of ICs in rat LSECs. Internalized ICs are degraded with slow kinetics, and IC internalization is not linked to receptor downregulation. After internalization, the receptor recycles to the cell surface both with and without ICs. Thus, FcgammaRIIb2 in rat LSECs is used as both a recycling receptor and a receptor for efficient IC clearance.

ABCA1, ABCG1 and SR-BI: hormonal regulation in primary rat hepatocytes and human cell lines.

BACKGROUND: Scavenger receptor type B class I (SR-BI), ABC transporter A1 (ABCA1) -and G1 (ABCG1) all play important roles in the reverse cholesterol transport. Reverse cholesterol transport is a mechanism whereby the body can eliminate excess cholesterol. Here, the regulation of SR-BI, ABCA1, and ABCG1 by dexamethasone (a synthetic glucocorticoid) and insulin were studied in order to gain more insight into the role of these two hormones in the cholesterol metabolism. RESULTS: By use of real time RT-PCR and Western blotting we examined the expression of our target genes. The results show that SR-BI, ABCA1 and ABCG1 mRNA expression increased in response to dexamethasone while insulin treatment reduced the expression in primary rat hepatocytes. The stimulatory effect of dexamethasone was reduced by the addition of the anti-glucocorticoid mifepristone. In HepG2 cells and THP-1 macrophages, however, the effect of dexamethasone was absent or inhibitory with no significant change in the presence of mifepristone. The latter observation may be a result of the low protein expression of glucocorticoid receptor (GR) in these cell lines. CONCLUSION: Our results illustrates that insulin and glucocorticoids, two hormones crucial in the carbohydrate metabolism, also play an important role in the regulation of genes central in reverse cholesterol transport. We found a marked difference in mRNA expression between the primary cells and the two established cell lines when studying the effect of dexamethasone which may result from the varying expression levels of GR.

Bile acids reduce SR-BI expression in hepatocytes by a pathway involving FXR/RXR, SHP, and LRH-1.

Hepatic SR-BI mediates uptake of circulating cholesterol into liver hepatocytes where a part of the cholesterol is metabolised to bile acids. In the hepatocytes, bile acids reduce their own synthesis by a negative feedback loop to prevent toxic high levels of bile acids. Bile acid-activated FXR/RXR represses expression of CYP7A1, the rate-limiting enzyme during bile acid synthesis, by inducing the expression of SHP, which inhibits LXR/RXR and LRH-1-transactivation of CYP7A1. The present paper presents data indicating that CDCA suppresses SR-BI expression by the same pathway. As previously reported, LRH-1 induces SR-BI promoter activity. Here we show that CDCA or over-expression of SHP inhibit this transactivation. No FXR-response element was identified in the bile acid-responsive region of the SR-BI promoter (-1200bp/-937bp). However, a binding site for LRH-1 was characterised and shown to specifically bind LRH-1. The present study shows that also the SR-BI-mediated supply of cholesterol, the substrate for bile acid synthesis, is feedback regulated by bile acids.

Pregnane X receptor-agonists down-regulate hepatic ATP-binding cassette transporter A1 and scavenger receptor class B type I.

Pregnane X receptor (PXR) is the molecular target for a wide variety of endogenous and xenobiotic compounds. It regulates the expression of genes central to the detoxification (cytochrome P-450 enzymes) and excretion (xenobiotic transporters) of potentially harmful compounds. The aim of the present investigation was to determine the role of PXR in regulation of high-density lipoprotein (HDL) cholesterol metabolism by studying its impact on ATP-binding cassette transporter A1 (ABCA1) and scavenger receptor class B type I (SR-BI) expression in hepatocytes. ABCA1 and SR-BI are major factors in the exchange of cholesterol between cells and HDL. Expression analyses were performed using Western blotting and quantitative real time RT-PCR. Luciferase reporter gene assays were used to measure promoter activities. Total cholesterol was measured enzymatically after lipid extraction (Folch's method). The expression of ABCA1 and SR-BI was inhibited by the PXR activators rifampicin and lithocholic acid (LCA) in HepG2 cells and pregnenolone 16alpha-carbonitrile (PCN) in primary rat hepatocytes. Thus, PXR appears to be a regulator of hepatic cholesterol transport by inhibiting genes central to cholesterol uptake (SR-BI) and efflux (ABCA1).

Uptake of denatured collagen into hepatic stellate cells: evidence for the involvement of urokinase plasminogen activator receptor-associated protein/Endo180.

Tissue remodelling is dependent on the integration of signals that control turnover of ECM (extracellular matrix). Breakdown and endocytosis of collagen, a major component of the ECM, is central to this process. Whereas controlled secretion of matrix-degrading enzymes (such as matrix metalloproteinases) has long been known to mediate ECM breakdown, it is becoming clear that uPARAP/Endo180 (where uPARAP stands for urokinase plasminogen activator receptor-associated protein) serves as a receptor that mediates endocytosis of collagen by several types of cells. In the liver, the stellate cells play a major role in turnover of ECM including collagens. These cells synthesize various collagens and also produce matrix metalloproteinases. In the present study, we investigated the capacity of rat hepatic stellate cells to endocytose and degrade 125I-labelled heat-denatured collagen I. It was found that the collagen is efficiently taken up and degraded by these cells. Degradation was inhibited by inhibitors of lysosomal proteases (leupeptin and E-64d) and the vacuolar proton pump (concanamycin A), indicating that it takes place in lysosomes. Furthermore, endocytosed FITC-labelled collagen was shown to reach late endocytic compartments in which it colocalized with LysoTracker (a marker of late endocytic compartments). Competition experiments showed that uPA and unlabelled collagen are capable of inhibiting binding and uptake of [125I]collagen in a dose-dependent manner. Moreover, Western-blot analysis of cell lysate (using a polyclonal rabbit human-Endo180 antiserum) revealed a single band at 180 kDa. In addition, the antiserum was capable of reducing [125I]collagen binding to the cell surface. Finally, using two primers designed from the human uPARAP/Endo180 mRNA sequence, the expression of uPARAP/Endo180 mRNA was detected by reverse transcriptase-PCR. These results together suggest that uPARAP/Endo180 mediates endocytosis of collagen in rat liver stellate cells.

Hepatic cellular distribution and degradation of iron oxide nanoparticles following single intravenous injection in rats: implications for magnetic resonance imaging.

The purpose of this study was to determine the cellular distribution and degradation in rat liver following intravenous injection of superparamagnetic iron oxide nanoparticles used for magnetic resonance imaging (NC100150 Injection). Relaxometric and spectrophotometric methods were used to determine the concentration of the iron oxide nanoparticles and their degradation products in isolated rat liver parenchymal, endothelial and Kupffer cell fractions. An isolated cell phantom was also constructed to quantify the effect of the degradation products on the loss of MR signal in terms of decreased transverse relaxation times, T2*. The results of this study show that iron oxide nanoparticles found in the NC100150 Injection were taken up and distributed equally in both liver endothelial and Kupffer cells following a single 5 mg Fe/kg body wt. bolus injection in rats. Whereas endothelial and Kupffer cells exhibited similar rates of uptake and degradation, liver parenchymal cells did not take up the NC100150 Injection iron oxide particles. Light-microscopy methods did, however, indicate an increased iron load, presumably as ferritin/hemosiderin, within the hepatocytes 24 h post injection. The study also confirmed that compartmentalisation of ferritin/hemosiderin may cause a significant decrease in the MRI signal intensity of the liver. In conclusion, the combined results of this study imply that the prolonged presence of breakdown product in the liver may cause a prolonged imaging effect (in terms of signal loss) for a time period that significantly exceeds the half-life of NC100150 Injection iron oxide nanoparticles in liver.

Intralobular Distribution of Vitamin A-Storing Lipid Droplets in Hepatic Stellate Cells with Special Reference to Polar Bear and Arctic Fox.

We examined the liver of adult polar bears, arctic foxes, and rats by gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, hematoxylin-eosin staining, staining with Masson's trichrome, Ishii and Ishii's silver impregnation, and transmission electron microscopical morphometry. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. These results indicate that the hepatic stellate cells of the polar bears and arctic foxes possess heterogeneity of vitamin A-storing capacity in their liver lobules.

Clathrin-dependent endocytosis.

The process by which clathrin-coated vesicles are produced involves interactions of multifunctional adaptor proteins with the plasma membrane, as well as with clathrin and several accessory proteins and phosphoinositides. Here we review recent findings highlighting new insights into mechanisms underlying clathrin-dependent endocytosis.

Hepatic scavenger receptor class B, type I is stimulated by peroxisome proliferator-activated receptor gamma and hepatocyte nuclear factor 4alpha.

Excessive cellular cholesterol is transported to the liver by a pathway called 'reverse cholesterol transport.' Scavenger receptor class B, type I (SR-BI) mediates cholesterol uptake in the liver. Polyunsaturated fatty acids, known to activate peroxisome proliferator-activated receptor (PPAR), have been reported to increase hepatic cholesterol uptake. We found in the present study that PPARgamma induces expression of SR-BI in rat hepatocytes, liver endothelial cells, and Kupffer cells. In contrast, PPARalpha increased SR-BI levels only in hepatocytes and liver endothelial cells. PPARgamma/RXR binds to a response element between -459 and -472 bp in the human SR-BI promoter. Furthermore, hepatocyte nuclear factor 4alpha (HNF4alpha) was found to enhance PPARgamma-mediated SR-BI transcription. Thiazolidinedione (TZD)-activated PPARgamma/RXR increased hepatic SR-BI levels, which may lead to increased hepatic cholesterol uptake and less accumulation of lipids in peripheral tissues. The present results are in agreement with previous reports, indicating that specific PPARgamma-agonists (such as TZDs) protect against atherosclerosis.

Phosphoinositide 3-kinase regulates maturation of lysosomes in rat hepatocytes.

To obtain information about the role of phosphoinositide 3-kinase (PI3K) in the endocytic pathway in hepatocytes, the uptake and intracellular transport of asialo-orosomucoid (ASOR) was followed in cells treated with wortmannin or LY294002. The two inhibitors, at concentrations known to inhibit the enzyme, did not affect internalization or the number of surface asialoglycoprotein receptors, but they caused a paradoxical increase (approx. 50% above control values) in the degradation of ASOR labelled with [(125)I]tyramine cellobiose ([(125)I]TC). Wortmannin or LY204002 inhibited the autophagic sequestration of lactate dehydrogenase very effectively, and the enhanced degradation of [(125)I]TC-ASOR could be an indirect effect of reduced autophagy, as an amino acid mixture known to inhibit autophagy also caused increased degradation of [(125)I]TC-ASOR, and its effect was not additive to that of wortmannin or LY294002. Wortmannin or LY294002 had pronounced effects on the late parts of the endocytic pathway in the hepatocytes: first, dense lysosomes disappeared and were replaced by swollen vesicles; secondly, degradation of [(125)I]TC-ASOR took place in an organelle of lower buoyant density (in a sucrose gradient) than the bulk of lysosomes (identified in the gradient by lysosomal marker enzymes). With increasing length of incubation with wortmannin or LY294002, the density distributions of the lysosomal markers also shifted to lower density and gradually approached that of the labelled degradation products. The labelled degradation products formed from [(125)I]TC-labelled proteins were trapped at the site of formation, because they did not penetrate the vesicle membranes. The results obtained indicate that internalization and intracellular transport of ASOR to lysomes may take place in the absence of PI3K activity in rat hepatocytes. On the other hand, fusion of late endosomes with lysosomes seems to produce 'hybrid organelles' (active lysosomes) that are unable to mature into dense lysosomes.

Oxysterol-activated LXRalpha/RXR induces hSR-BI-promoter activity in hepatoma cells and preadipocytes.

SR-BI mediates exchange of cholesterol between HDL and cells, and is a crucial factor in the transport of excessive cellular cholesterol from extrahepatic tissues to the liver ("reverse cholesterol transport") and, therefore, also for cholesterol homeostasis. Hepatic SR-BI mediates transfer of HDL-cholesterol to the hepatocytes where cholesterol may be metabolised to bile acids. LXR and SREBP are key factors in the regulation of cholesterol metabolism. The purpose of the present study was to determine whether these transcription factors are involved in the regulation of SR-BI. Here we show that LXRalpha/RXR and LXRbeta/RXR induce SR-BI transcription in human and murine hepatoma cell lines, and in 3T3-L1 preadipocytes independently of SREBP-1. The LXR/RXR response was mapped within -1,200 to -937 of the promoter region. Gel mobility shift analysis confirmed that the putative LXR response element bound LXRalpha/RXR and LXRbeta/RXR heterodimers.

Metabolism of oxidized and chemically modified low density lipoproteins in rainbow trout--clearance via scavenger receptors.

Oxidative modifications of low density lipoprotein (LDL) convert LDL into a ligand recognized by a variety of scavenger receptors (SR) in mammals. This oxidized LDL (oxLDL) activate several cell types, and have been shown to induce expression of a variety of genes in mammals. Lipoproteins of poikilothermic animals like salmonid fishes contain high levels of polyunsaturated fatty acids susceptible to oxidative modifications. We have investigated, and found trout LDL to be susceptible to oxidation in the presence of Cu(2+). When oxidized or acetylated trout LDL was injected intravenously, the clearance rate was increased compared to that of native LDL. Modified LDL was taken up almost exclusively in the kidney, whereas native LDL was also taken up in the liver. Uptake of both oxLDL and acetylated LDL in the kidney was significantly inhibited by lipoteichoic acid (LTA) and formaldehyde treated BSA (fBSA), both of which are known ligands of SR.