The Relationship between Fenestrations,Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished.     

Angiogenesis activators and inhibitors differentially regulate caveolin-1 expression and caveolae formation in vascular endothelial cells. Angiogenesis inhibitors block vascular endothelial growth factor-induced down-regulation of caveolin-1.

Angiogenesis is the process by which new blood vessels are formed via proliferation of vascular endothelial cells. A variety of angiogenesis inhibitors that antagonize the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have recently been identified. However, the mechanism by which these diverse angiogenesis inhibitors exert their common effects remains largely unknown. Caveolin-1 and -2 are known to be highly expressed in vascular endothelial cells both in vitro and in vivo. Here, we examine the potential role of caveolins in the angiogenic response. For this purpose, we used the well established human umbilical vein endothelial cell line, ECV 304. Treatment of ECV 304 cells with known angiogenic growth factors (VEGF, bFGF, or hepatocyte growth factor/scatter factor), resulted in a dramatic reduction in the expression of caveolin-1. This down-regulation event was selective for caveolin-1, as caveolin-2 levels remained constant under these conditions of growth factor stimulation. VEGF-induced down-regulation of caveolin-1 expression also resulted in the morphological loss of cell surface caveolae organelles as seen by transmission electron microscopy. A variety of well characterized angiogenesis inhibitors (including angiostatin, fumagillin, 2-methoxy estradiol, transforming growth factor-beta, and thalidomide) effectively blocked VEGF-induced down-regulation of caveolin-1 as seen by immunoblotting and immunofluorescence microscopy. However, treatment with angiogenesis inhibitors alone did not significantly affect the expression of caveolin-1. PD98059, a specific inhibitor of mitogen-activated protein kinase and a known angiogenesis inhibitor, also blocked the observed VEGF-induced down-regulation of caveolin-1. Furthermore, we show that caveolin-1 can function as a negative regulator of VEGF-R (KDR) signal transduction in vivo. Thus, down-regulation of caveolin-1 may be an important step along the pathway toward endothelial cell proliferation.     

Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro

Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.     

Dengue virus infection of SK Hep1 cells: inhibition of in vitro angiogenesis and altered cytomorphology by expressed viral envelope glycoprotein

Dengue virus (DENV) infection of human endothelial cells has been implicated in the pathobiology of dengue hemorrhagic fever and dengue shock syndrome. However, the mechanisms by which DENV infections alter the functional physiology of endothelial cells remain incompletely understood. In the present study, we examined the susceptibility of a human liver sinusoidal endothelial cell line SK Hep1 to all four serotypes of DENV and studied the effect of the virus on in vitro angiogenesis. All four serotypes of DENV could infect the SK Hep1 cells, but showed variable cytopathic effects, the most pronounced being that of DENV-2. Electron microscopy of the infected cells showed significant ultrastructural changes. In vitro angiogenesis assays on DENV-2 exposed SK Hep1 cells in the matrigel system showed inhibition compared with the controls. Importantly, transfection and transient expression of the DENV-2 envelope glycoprotein (E) in these cells showed drastic alterations in cell shapes and the E protein could be localized by fluorescence microscopy in terminal knob-like structures. Therefore, SK Hep1, a human hepatic sinusoid-derived endothelial cell line, may constitute a potential model to study DENV-endothelial cell interactions in vitro, especially towards understanding the possible virus-induced changes in hepatic endothelium and its role in disease pathogenesis.     

The human hyaluronan receptor for endocytosis (HARE/Stabilin-2) is a systemic clearance receptor for heparin

The hyaluronic acid receptor for endocytosis (HARE; also designated Stabilin-2) mediates systemic clearance of hyaluronan and chondroitin sulfates from the vascular and lymphatic circulations. The internalized glycosaminoglycans are degraded in lysosomes, thus completing their normal turnover process. Sinusoidal endothelial cells of human liver, lymph node, and spleen express two HARE isoforms of 315 and 190 kDa. Here we report that the 190- and 315-kDa HARE isoforms, expressed stably either in Flp-In 293 cell lines or as soluble ectodomains, specifically bind heparin (Hep). The K(d) for Hep binding to purified 190- and 315-kDa HARE ectodomains was 17.2 +/- 4.9 and 23.4 +/- 5.3 nm, respectively. Cells expressing HARE readily and specifically internalized (125)I-streptavidin-biotin-Hep complexes, which was inhibited >70% by hyperosmolar conditions, confirming that uptake is mediated by the clathrin-coated pit pathway. Internalization of Hep occurred for many hours with an estimated HARE recycling time of approximately 12 min. Internalized fluorescent streptavidin-biotin-Hep was present in a typical endocytic vesicular pattern and was delivered to lysosomes. We conclude that HARE in the sinusoidal endothelial cells of lymph nodes and liver likely mediates the efficient systemic clearance of Hep and many different Hep-binding protein complexes from the lymphatic and vascular circulations.     

Culture and characterization of sinusoidal endothelial cells isolated from human liver

Summary Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.     

Common lymphatic endothelial and vascular endothelial receptor-1 mediates the transmigration of regulatory T cells across human hepatic sinusoidal endothelium

The common lymphatic endothelial and vascular endothelial receptor (CLEVER-1; also known as FEEL-1 and stabilin-1) is a recycling and intracellular trafficking receptor with multifunctional properties. In this study, we demonstrate increased endothelial expression of CLEVER-1/stabilin-1 at sites of leukocyte recruitment to the inflamed human liver including sinusoids, septal vessels, and lymphoid follicles in inflammatory liver disease and tumor-associated vessels in hepatocellular carcinoma. We used primary cultures of human hepatic sinusoidal endothelial cells (HSEC) to demonstrate that CLEVER-1/stabilin-1 expression is enhanced by hepatocyte growth factor but not by classical proinflammatory cytokines. We then showed that CLEVER-1/stabilin-1 supports T cell transendothelial migration across HSEC under conditions of flow with strong preferential activity for CD4 FoxP3(+) regulatory T cells (Tregs). CLEVER-1/stabilin-1 inhibition reduced Treg transendothelial migration by 40% and when combined with blockade of ICAM-1 and vascular adhesion protein-1 (VAP-1) reduced it by >80%. Confocal microscopy demonstrated that 60% of transmigrating Tregs underwent transcellular migration through HSEC via ICAM-1- and VAP-1-rich transcellular pores in close association with CLEVER-1/stabilin-1. Thus, CLEVER-1/stabilin-1 and VAP-1 may provide an organ-specific signal for Treg recruitment to the inflamed liver and to hepatocellular carcinoma.     

Loss of caveolin-1 polarity impedes endothelial cell polarization and directional movement

The ability of a cell to move requires the asymmetrical organization of cellular activities. To investigate polarized cellular activity in moving endothelial cells, human endothelial cells were incubated in a Dunn chamber to allow migration toward vascular endothelial growth factor. Immunofluorescent staining with a specific antibody against caveolin-1 revealed that caveolin-1 was concentrated at the rear of moving cells. Similarly, monolayer scraping to induce random cell walk resulted in relocation of caveolin-1 to the cell rear. These results suggest that posterior polarization of caveolin-1 is a common feature both for chemotaxis and chemokinesis. Dual immunofluorescent labeling showed that, during cell spreading, caveolin-1 was compacted in the cell center and excluded from nascent focal contacts along the circular lamellipodium, as revealed by integrin beta1 and FAK staining. When cells were migrating, integrin beta1 and FAK appeared at polarized lamellipodia, whereas caveolin-1 was found at the posterior of moving cells. Notably, wherever caveolin-1 was polarized, there was a conspicuous absence of lamellipod protrusion. Transmission electron microscopy showed that caveolae, similar to their marker caveolin-1, were located at the cell center during cell spreading or at the cell rear during cell migration. In contrast to its unphosphorylated form, tyrosine-phosphorylated caveolin-1, upon fibronectin stimulation, was associated with the focal complex molecule phosphopaxillin along the lamellipodia of moving cells. Thus, unphosphorylated and phosphorylated caveolin-1 were located at opposite poles during cell migration. Importantly, loss of caveolin-1 polarity by targeted down-regulation of the protein prevented cell polarization and directional movement. Our present results suggest a potential role of caveolin polarity in lamellipod extension and cell migration.     

VEGF can act as vascular permeability factor in the hepatic sinusoids through upregulation of porosity of endothelial cells

VEGF is shown to be a vascular permeability factor (VPF) as well as a growth stimulatory factor on endothelial cells. In the hepatic sinusoids, endothelial cells express flt-1 and KDR/flk-1, receptors for VEGF. These cells, in primary culture, proliferate in response to VEGF stimulation. However, the role of VEGF as VPF in the hepatic sinusoids is to be elucidated. The effect of VEGF on the porosity of sinusoidal endothelial cells was studied. Sinusoidal endothelial cells were isolated from rats and cultured in DMEM containing 10% FCS on plastic dishes coated with type I collagen for 16 and 48 h for morphological examination and cell-number measurement, respectively. When the cells were cultured without VEGF addition, their number was decreased at 48 h compared to that at 16 h. However, the number was unchanged in the cells cultured with VEGF at 10 ng/mL and increased with addition of VEGF at 100 ng/mL. Scanning electron microscopic examination revealed that sieve-plate appearance of the cells was impaired in culture with no VEGF addition, but the appearance was maintained in culture with VEGF at 10 ng/mL or more. The cells cultured with VEGF at 100 ng/mL showed significantly increased number and size of pores compared to the cells cultured with VEGF at 10 ng/mL, suggesting that sinusoidal endothelial cells proliferating in response to VEGF may increase their porosity. It is concluded that VEGF can act as VPF in the hepatic sinusoids through regulation of endothelial cell porosity.     

Induction of LYVE-1/stabilin-2-positive liver sinusoidal endothelial-like cells from embryoid bodies by modulation of adrenomedullin-RAMP2 signaling

Embryonic stem cells (ESCs) are a useful source for various cell lineages. So far, however, progress toward reconstitution of mature liver morphology and function has been limited. We have shown that knockout mice deficient in adrenomedullin (AM), a multifunctional endogenous peptide, or its receptor-activity modifying protein (RAMP2) die in utero due to poor vascular development and hemorrhage within the liver. In this study, using embryoid bodies (EBs)-culture system, we successfully induced liver sinusoidal endothelial-like cells by modulation of AM-RAMP2. In an EB differentiation system, we found that co-administration of AM and SB431542, an inhibitor of transforming growth factor β (TGFβ) receptor type 1, markedly enhanced differentiation of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1)/stabilin-2-positive endothelial cells. These cells showed robust endocytosis of acetylated low-density lipoprotein (Ac-LDL) and upregulated expression of liver sinusoidal endothelial cells (LSECs)-specific markers, including factor 8 (F8), Fc-γ receptor 2b (Fcgr2b), and mannose receptor C type 1 (Mrc1), and also possessed fenestrae-like structure, a key morphological feature of LSECs. In RAMP2-null liver, by contrast, LYVE-1 was downregulated in LSECs, and the sinusoidal structure was disrupted. Our findings highlight the importance of AM-RAMP2 signaling for development of LSECs.     

Filtration effect of endothelial fenestrations on chylomicron transport in neonatal rat liver sinusoids

Summary The function of endothelial fenestrations in the control of the passage of chylomicrons from the blood to the liver parenchymal cells was studied ultrastructurally in neonatal rats. Measurement of circulating chylomicrons and endothelial fenestrations as well as chylomicrons in the space of Disse at 2–20 h after the onset of suckling revealed a substantial filtration effect of endothelial fenestrations on chylomicrons larger than 250 nm in diameter. Direct uptake of chylomicrons by parenchymal cells was observed to occur by pinocytosis; Kupffer cells endocytosed only very few chylomicrons, and endothelial cells were inactive in this respect.     

Mice transgenic for IL-1 receptor antagonist protein are resistant to herpetic stromal keratitis: possible role for IL-1 in herpetic stromal keratitis pathogenesis

Ocular infection with HSV may result in the blinding immunoinflammatory lesion stromal keratitis (SK). This represents a CD4+ T cell-mediated immunopathologic lesion in both humans and a mouse model. Early events in the pathogenesis that set the stage for SK are poorly understood. The present study evaluates the role of IL-1 using a transgenic mouse that overexpresses the IL-1 receptor antagonist (IL-1ra) protein. Such transgenic mice were markedly resistant to SK compared with IL-1ra(-/-) and C57BL/6 control animals. The resistance was shown to be the consequence of reduced expression of molecules such as IL-6, macrophage-inflammatory protein-2, and vascular endothelial growth factor, normally up-regulated directly or indirectly by IL-1. A critical event impaired in IL-1ra transgenic mice was vascular endothelial growth factor production with a consequent marked reduction in angiogenesis, an essential step in SK pathogenesis. Targeting IL-1 could prove to be a worthwhile therapeutic approach to control SK, an important cause of human blindness.     

Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. eNOS activation by sphingosine 1-phosphate and the role of caveolin-1 in sphingolipid signal transduction

Plasmalemmal caveolae are membrane microdomains that are specifically enriched in sphingolipids and contain a wide array of signaling proteins, including the endothelial isoform of nitric-oxide synthase (eNOS). EDG-1 is a G protein-coupled receptor for sphingosine 1-phosphate (S1P) that is expressed in endothelial cells and has been implicated in diverse vascular signal transduction pathways. We analyzed the subcellular distribution of EDG-1 in COS-7 cells transiently transfected with cDNA constructs encoding epitope-tagged EDG-1. Subcellular fractionation of cell lysates resolved by ultracentrifugation in discontinuous sucrose gradients revealed that approximately 55% of the EDG-1 protein was recovered in fractions enriched in caveolin-1, a resident protein of caveolae. Co-immunoprecipitation experiments showed that EDG-1 could be specifically precipitated by antibodies directed against caveolin-1 and vice versa. The targeting of EDG-1 to caveolae-enriched fractions was markedly increased (from 51 +/- 11% to 93 +/- 14%) by treatment of transfected cells with S1P (5 microm, 60 min). In co-transfection experiments expressing EDG-1 and eNOS cDNAs in COS-7 cells, we found that S1P treatment significantly and specifically increased nitric-oxide synthase activity, with an EC(50) of 30 nm S1P. Overexpression of transfected caveolin-1 cDNA together with EDG-1 and eNOS markedly diminished S1P-mediated eNOS activation; caveolin overexpression also attenuated agonist-induced phosphorylation of EDG-1 receptor by >90%. These results suggest that the interaction of the EDG-1 receptor with caveolin may serve to inhibit signaling through the S1P pathway, even as the targeting of EDG-1 to caveolae facilitates the interactions of this receptor with ligands and effectors that are also targeted to caveolae. The agonist-modulated targeting of EDG-1 to caveolae and its dynamic inhibitory interactions with caveolin identify new points for regulation of sphingolipid-dependent signaling in the vascular wall.     

Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells

Caveolin-1 is a scaffolding/regulatory protein that interacts with diverse signaling molecules in endothelial cells. To explore the role of this protein in receptor-modulated signaling pathways, we transfected bovine aortic endothelial cells (BAEC) with small interfering RNA (siRNA) duplexes to down-regulate caveolin-1 expression. Transfection of BAEC with duplex siRNA targeted against caveolin-1 mRNA selectively "knocked-down" the expression of caveolin-1 by approximately 90%, as demonstrated by immunoblot analyses of BAEC lysates. We used discontinuous sucrose gradients to purify caveolin-containing lipid rafts from siRNA-treated endothelial cells. Despite the near-total down-regulation of caveolin-1 expression, the lipid raft targeting of diverse signaling proteins (including the endothelial isoform of nitric-oxide synthase, Src-family tyrosine kinases, Galphaq and the insulin receptor) was unchanged. We explored the consequences of caveolin-1 knockdown on kinase pathways modulated by the agonists sphingosine-1 phosphate (S1P) and vascular endothelial growth factor (VEGF). siRNA-mediated caveolin-1 knockdown enhanced basal as well as S1P- and VEGF-induced phosphorylation of the protein kinase Akt and did not modify the basal or agonist-induced phosphorylation of extracellular signal-regulated kinases 1/2. Caveolin-1 knock-down also significantly enhanced the basal and agonist-induced activity of the small GTPase Rac. We used siRNA to down-regulate Rac expression in BAEC, and we observed that Rac knockdown significantly reduced basal, S1P-, and VEGF-induced Akt phosphorylation, suggesting a role for Rac activation in the caveolin siRNA-mediated increase in Akt phosphorylation. By using siRNA to knockdown caveolin-1 and Rac expression in cultured endothelial cells, we have found that caveolin-1 does not seem to be required for the targeting of signaling molecules to caveolae/lipid rafts and that caveolin-1 differentially modulates specific kinase pathways in endothelial cells.     

Caveolin-1 and caveolae in atherosclerosis: differential roles in fatty streak formation and neointimal hyperplasia

PURPOSE OF REVIEW: Caveolae are 50-100 nm cell surface plasma membrane invaginations observed in terminally differentiated cells. They are characterized by the presence of the protein marker caveolin-1. Caveolae and caveolin-1 are present in almost every cell type that has been implicated in the development of an atheroma. These include endothelial cells, macrophages, and smooth muscle cells. Caveolae and caveolin-1 are involved in regulating several signal transduction pathways and processes that play an important role in atherosclerosis. RECENT FINDINGS: Several recent studies using genetically engineered mice (Cav-1 (-/-) null animals) have now clearly demonstrated a role for caveolin-1 and caveolae in the development of atherosclerosis. In fact, they suggest a rather complex one, either proatherogenic or antiatherogenic, depending on the cell type examined. For example, in endothelial cells, caveolin-1 and caveolae may play a proatherogenic role by promoting the transcytosis of LDL-cholesterol particles from the blood to the sub-endothelial space. In contrast, in smooth muscle cells, the ability of caveolin-1 to negatively regulate cell proliferation (neointimal hyperplasia) may have an antiatherogenic effect. SUMMARY: Caveolin-1 and caveolae play an important role in several steps involved in the initiation of an atheroma. Development of new drugs that regulate caveolin-1 expression may be important in the prevention or treatment of atherosclerotic vascular disease.     

Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.

Caveolin-1 is the principal structural protein of caveolae membranes in fibroblasts and endothelia. Recently, we have shown that the human CAV-1 gene is localized to a suspected tumor suppressor locus, and mutations in Cav-1 have been implicated in human cancer. Here, we created a caveolin-1 null (CAV-1 -/-) mouse model, using standard homologous recombination techniques, to assess the role of caveolin-1 in caveolae biogenesis, endocytosis, cell proliferation, and endothelial nitric-oxide synthase (eNOS) signaling. Surprisingly, Cav-1 null mice are viable. We show that these mice lack caveolin-1 protein expression and plasmalemmal caveolae. In addition, analysis of cultured fibroblasts from Cav-1 null embryos reveals the following: (i) a loss of caveolin-2 protein expression; (ii) defects in the endocytosis of a known caveolar ligand, i.e. fluorescein isothiocyanate-albumin; and (iii) a hyperproliferative phenotype. Importantly, these phenotypic changes are reversed by recombinant expression of the caveolin-1 cDNA. Furthermore, examination of the lung parenchyma (an endothelial-rich tissue) shows hypercellularity with thickened alveolar septa and an increase in the number of vascular endothelial growth factor receptor (Flk-1)-positive endothelial cells. As predicted, endothelial cells from Cav-1 null mice lack caveolae membranes. Finally, we examined eNOS signaling by measuring the physiological response of aortic rings to various stimuli. Our results indicate that eNOS activity is up-regulated in Cav-1 null animals, and this activity can be blunted by using a specific NOS inhibitor, nitro-l-arginine methyl ester. These findings are in accordance with previous in vitro studies showing that caveolin-1 is an endogenous inhibitor of eNOS. Thus, caveolin-1 expression is required to stabilize the caveolin-2 protein product, to mediate the caveolar endocytosis of specific ligands, to negatively regulate the proliferation of certain cell types, and to provide tonic inhibition of eNOS activity in endothelial cells.     

Oxidative stress disrupts nitric oxide synthase activation in liver endothelial cells

Oxidative stress may mediate vascular disruption associated with a loss of endothelial nitric oxide synthase (eNOS) activity and a hypersensitivity to the constrictor effects of endothelin-1 (ET-1). We hypothesize that this is due, in part, to uncoupling of ET(B) receptors from eNOS activation. Thus, we tested whether oxidative stress (OS) affects liver vascular relaxation by reducing basal and ET-1-induced NO production. Primary sinusoidal endothelial cell cultures were pretreated with H(2)O(2) (25 microM) for 1 or 6 h before a 10-min ET-1 stimulation. OS resulted in a significant basal and ET-1-induced decrease in NO production. Acute OS increased the monomeric form of the inhibitory protein caveolin-1 (1.2 +/- 0.05 vs 0.9 +/- 0.02, p < 0.01) and increased the eNOS-caveolin association as determined by coimmunoprecipitation (1.24 +/- 0.04 vs 0.97 +/- 0.04, p < 0.05). ET-1 stimulation further exacerbated these effects. Subacute OS inhibited ET-1-induced eNOS phosphorylation of serine 1177 (activation residue) (1 +/- 0.07 vs 1.6 +/- 0.04, p < 0.05) and dephosphorylation of the inhibitory residue threonine 495 (1.5 +/- 0.08 vs 0.7 +/- 0.02, p < 0.01). Additionally subacute OS resulted in dissociation of eNOS from ET(B) (0.8 +/- 0.09 vs 1.2 +/- 0.06, p < 0.05). Our findings indicate that acute and subacute oxidative stress result in the inhibition of induced nitric oxide synthase activity through distinct mechanisms dependent on caveolin-1 inhibition, ET(B) dissociation, and eNOS phosphorylation.