Caveolae In Endothelial Cells Research Articles

Non-nuclear Estrogen Receptor Signaling in the Endothelium

In addition to the classical function of estrogen receptors (ER) as transcription factors, evidence continues to accumulate that they mediate non-nuclear processes in numerous cell types, including the endothelium, in which they activate endothelial NO synthase. Non-nuclear ER signaling entails unique post-translational modifications and protein-protein interactions of the receptor with adaptor molecules, kinases, and G proteins. Recent in vitro and in vivo studies in mice using an estrogen-dendrimer conjugate that is excluded from the nucleus indicate that non-nuclear ER activation underlies the migration and growth responses of endothelial cells to estrogen but not the growth responses of endometrial or breast cancer cells to the hormone. In this minireview, the features of ERα and protein-protein interactions that enable it to invoke extranuclear signaling in the endothelium and the consequences of that signaling are discussed.

Epigallocatechin-gallate stimulates NF-E2-related factor and heme oxygenase-1 via caveolin-1 displacement

Flavonoids, such as the tea catechin epigallocatechin-gallate (EGCG), can protect against atherosclerosis by decreasing vascular endothelial cell inflammation. Heme oxygenase-1 (HO-1) is an enzyme that plays an important role in vascular physiology, and its induction may provide protection against atherosclerosis. Heme oxygenase-1 can be compartmentalized in caveolae in endothelial cells. Caveolae are plasma microdomains important in vesicular transport and the regulation of signaling pathways associated with the pathology of vascular diseases. We hypothesize that caveolae play a role in the uptake and transport of EGCG and mechanisms associated with the anti-inflammatory properties of this flavonoid. To test this hypothesis, we explored the effect of EGCG on the induction of NF-E2-related factor (Nrf2) and HO-1 in endothelial cells with or without functional caveolae. Treatment with EGCG activated Nrf2 and increased HO-1 expression and cellular production of bilirubin. In addition, EGCG rapidly accumulated in caveolae, which was associated with caveolin-1 displacement from the plasma membrane towards the cytosol. Similar to EGCG treatment, silencing of caveolin-1 by siRNA technique also resulted in up-regulation of Nrf2, HO-1 and bilirubin production. These data suggest that EGCG-induced caveolin-1 displacement may reduce endothelial inflammation.

Activity of BKCa Channel Is Modulated by Membrane Cholesterol Content and Association with Na+/K+-ATPase in Human Melanoma IGR39 Cells

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.

Oxidative stress-induced endothelial cell damage in thyroidectomized rat

Patients with hypothyroidism are considered to have an increased risk of developing atherosclerosis. Uncoupling of endothelial nitric oxide synthase from tetrahyrobiopterin, an essential cofactor, leads to the decrease of nitric oxide production and increase in reactive oxygen species. Both mechanisms contribute to atherosclerotic vascular disease. The aim of this study was to investigate whether hypothyroidism influences the systemic redox-state and the structure of aortic vascular endothelium in thyroidectomized rats. Twenty-eight Sprague-Dawley rats were randomly assigned to two groups; sham thyroidectomy and near-total thyroidectomy. Three weeks after surgery, nitric oxide in plasma, homocysteine, cysteine, glutathione levels and superoxide dismutase activity were measured in serum, and biopterine and creatinine were measured in urine. Aortic endothelium samples were processed for light and transmission electron microscopy. Although superoxide dismutase activity remained constant, urine biopterin levels and serum nitric oxide levels decreased in hypothyroid rats compared to their euthyroid controls. Aortic endothelium showed early features of atherosclerosis, and transmission electron microscopy revealed budding of caveolae in endothelial cells. It is concluded that hypothyroidism might lead to systemic oxidative stress with resultant endothelial dysfunction, and subsequent occurrence of atherosclerosis.

Estradiol-17β-Induced Temporal and Spatial Partitioning of eNOS and Estrogen Receptors in Ovine Uterine Artery Endothelial Cells from Pregnant Sheep.

Pregnancy-induced elevations in uterine blood flow are closely correlated with concurrent increases in plasma estradiol-17β levels and the expression/activity of eNOS. Indeed during late ovine gestation a physiologic cause and effect relationship has been established in vivo in that unilateral blockade of estrogen receptors (w/ICI 182,780) or nitric oxide synthase (w/L-NAME) locally and by a similar magnitude reduces uteroplacental blood flow. Estradiol-17β binds to estrogen receptors alpha and beta (ERα/β) and binding to these receptors results in either rapid effects (non-classical, non-genomic) and/or chronic effects (classical-genomic). Interestingly, numerous proteins that are involved in the estradiol-17β-induced rapid eNOS activation and nitric oxide production are located in specialized membrane domains called the caveolae of uterine artery endothelial cells (UAECs). Furthermore, treatment with the calcium mobilizing agonist ATP induced rapid eNOS activation which is accompanied by eNOS repartitioning with alterations in its multi-site phosphorylation state. We hypothesized that estradiol-17β induces eNOS activation via the non-classical ER receptor and initiates the sequential and coordinated re-partitioning and activation of eNOS with altered multi-site phosphorylation. UAECs were isolated from uterine arteries of pregnant ewes (gestational day 120-130; term 147d). Confluent passage 4 UAECs were treated with vehicle (Control) or with estradiol-17β (10 nM) for 10 min. The caveolae were fractionated from UAECs using sucrose density gradient centrifugation, and immunoblotting was utilized to study the multi-site phosphorylation state of eNOS relative to the domain specific levels of ERs. In control UAECs, total eNOS was predominantly located in the caveolar domain, whereas it was detectable both in the caveolar and non-caveolar domains in estradiol-17β-treated cells. In control UAECs, stimulatory P635eNOS and P1177eNOS were both not detected in any of the fractions whereas inhibitory P114eNOS was detected strictly in the non-caveolar domain. In response to estradiol-17β, stimulatory P635eNOS was detected in all cellular domains whereas stimulatory P1177eNOS was detected mainly in the caveolar domain and barely visible in the non-caveolar domain. The inhibitory P114eNOS was decreased by estrogen to low levels in the non-caveolar domain and was not detected in the caveolar domain. In control UAECs, ERs were detected in a higher abundance in the non-caveolar domain compared to the caveolar domain. estradiol-17β did not alter the distribution of the ERs. Estradiol-17β produces temporal and spatial re-partitioning of eNOS from the caveolar to non-caveolar domains and alters its multi-site phosphorylation state and their distributions in UAECs. Furthermore, since estradiol-17β actions are not calcium mediated, the striking similarities of these results to the ATP-responses illustrate alternative activation pathways of calcium mobilizing agonists. These findings may be critical to understand estradiol-17β-induced gestational vascular adaptations. NIH HL49210, HD38843, HL87144, HL70562, R25GM083252. (platform)

Endothelial-Specific Overexpression of Caveolin-1 Accelerates Atherosclerosis in Apolipoprotein E-Deficient Mice

Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Genetic ablation of Cav-1 on an apolipoprotein E knockout background inhibits the progression of atherosclerosis, whereas re-expression of Cav-1 in the endothelium promotes lesion expansion. Although Cav-1-null mice are useful to delineate the importance of caveolae in atherosclerosis, there are additional problems that are difficult to dissect because loss of Cav-1 abolishes both the caveolae organelle as well as the Cav-1-mediated signaling pathways. To study how Cav-1 influences the progression of atherosclerosis in mice with caveolae, we generated a transgenic mouse that overexpresses Cav-1 in the endothelial cells in an apolipoprotein E-deficient background. We found that endothelial-specific overexpression of Cav-1 enhanced the progression of atherosclerosis in mice. Mechanistically, overexpression of Cav-1 reduced endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1 in vivo.

P391 OSTEOPONTIN GENE VARIATION AND CARDIO/CEREBROVASCULAR DISEASE PHENOTYPES

Caveolin-1 (Cav-1) is the major structural protein essential to the formation of the caveolae in endothelial cells. Genetic ablation of Cav-1 on an apolipoprotein E knockout background inhibits the progression of atherosclerosis, whereas re-expression of Cav-1 in the endothelium promotes lesion expansion. Although Cav-1-null mice are useful to delineate the importance of caveolae in atherosclerosis, there are additional problems that are difficult to dissect because loss of Cav-1 abolishes both the caveolae organelle as well as the Cav-1-mediated signaling pathways. To study how Cav-1 influences the progression of atherosclerosis in mice with caveolae, we generated a transgenic mouse that overexpresses Cav-1 in the endothelial cells in an apolipoprotein E-deficient background. We found that endothelial-specific overexpression of Cav-1 enhanced the progression of atherosclerosis in mice. Mechanistically, overexpression of Cav-1 reduced endothelial cell proliferation, migration, and nitric oxide production in vitro and increased expression of vascular cell adhesion molecule-1 in vivo.

Platelet-activating factor reduces endothelial nitric oxide production: role of acid sphingomyelinase

Platelet-activating factor (PAF) is a mediator of pulmonary oedema in acute lung injury that increases vascular permeability within minutes, partly through activation of acid sphingomyelinase (ASM). Since caveolae are rich in sphingomyelin and caveolin-1, which block endothelial nitric oxide (NO) synthase (eNOS) by direct binding, we examined the relationship between ASM, caveolin-1 and eNOS activity in the regulation of vascular permeability by PAF. In caveolar fractions from pulmonary vascular endothelial cells (isolated from perfused rat lungs) the abundance of caveolin-1 and eNOS increased rapidly after PAF perfusion. PAF treatment decreased endothelial NO (eNO) formation as assessed by in situ fluorescence microscopy. Restoration of eNO levels with PAPA-NONOate ((Z)-1-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium-1,2-diolate) mitigated the PAF-induced oedema. PAF treatment increased the ASM activity in caveolar fractions and perfusion with ASM decreased eNO production. Pharmacological inhibition of the ASM pathway with imipramine, D609 or dexamethasone blocked the PAF-induced increase of caveolin-1 and eNOS in caveolae, and the decrease in eNO production and oedema formation. We conclude that PAF causes ASM-dependent enrichment of caveolin-1 in caveolae of endothelial cells, leading to decreased eNO production which contributes to pulmonary oedema formation. These findings suggest rapid reduction in eNO production as a novel mechanism in the regulation of vascular permeability.

Decreased number of caveolae in endothelial cells impairs the relaxation induced by acetylcholine in hypertensive rat aortas

The present study was designed to investigate the contribution of endothelial cell caveolae to vascular relaxation in aortas from a normotensive (2K) and renal hypertensive (2K-1C) rat. For that purpose, concentration–effect curves to acetylcholine were constructed in 2K and 2K-1C intact endothelium aortic rings, in the absence or in the presence of the caveolae disassembler methyl-β-ciclodextrin. The potency (pD 2) and the maximum relaxant effect to acetylcholine were greater in 2K than in 2K-1C aortas. Methyl-β-ciclodextrin reduced the pD 2 in 2K and the maximum relaxant effect in both 2K and 2K-1C. The quantification of the caveolae number by electronic microscopy has shown a larger number of caveolae in 2K than in 2K-1C endothelial cells, which was reduced by methyl-β-ciclodextrin in both 2K and 2K-1C. The production of NO stimulated with acetylcholine was greater in 2K than in 2K-1C endothelial cells, and this effect was impaired by methyl-β-ciclodextrin in both 2K and 2K-1C. The cytosolic Ca 2+ concentration ([Ca 2+]c) was simultaneously measured in endothelial and smooth muscle cells stimulated with acetylcholine by confocal image of aortic slices. Acetylcholine produced a greater [Ca 2+]c increase in 2K than in 2K-1C endothelial cells, which response was inhibited by methyl-β-ciclodextrin only in 2K cells. In smooth muscle cells the reduction of [Ca 2+]c was higher in 2K than in 2K-1C. This effect was inhibited by methyl-β-ciclodextrin only in 2K cells. Taken together, our results suggest that the decreased number of caveolae in the endothelial cells from 2K-1C rat aortas is involved in the impaired effect of acetylcholine on [Ca 2+]c and NO.

Vascular Permeability and Pathological Angiogenesis in Caveolin-1-Null Mice

Caveolin-1, the signature protein of endothelial cell caveolae, has many important functions in vascular cells. Caveolae are thought to be the transcellular pathway by which plasma proteins cross normal capillary endothelium, but, unexpectedly, cav-1(-/-) mice, which lack caveolae, have increased permeability to plasma albumin. The acute increase in vascular permeability induced by agents such as vascular endothelial growth factor (VEGF)-A occurs through venules, not capillaries, and particularly through the vesiculo-vacuolar organelle (VVO), a unique structure composed of numerous interconnecting vesicles and vacuoles that together span the venular endothelium from lumen to ablumen. Furthermore, the hyperpermeable blood vessels found in pathological angiogenesis, mother vessels, are derived from venules. The present experiments made use of cav-1(-/-) mice to investigate the relationship between caveolae and VVOs and the roles of caveolin-1 in VVO structure in the acute vascular hyperpermeability induced by VEGF-A and in pathological angiogenesis and associated chronic vascular hyperpermeability. We found that VVOs expressed caveolin-1 variably but, in contrast to caveolae, were present in normal numbers and with apparently unaltered structure in cav-1(-/-) mice. Nonetheless, VEGF-A-induced hyperpermeability was strikingly reduced in cav-1(-/-) mice, as was pathological angiogenesis and associated chronic vascular hyperpermeability, whether induced by VEGF-A(164) or by a tumor. Thus, caveolin-1 is not necessary for VVO structure but may have important roles in regulating VVO function in acute vascular hyperpermeability and angiogenesis.

Nitric Oxide Release in Human Aortic Endothelial Cells Mediated by Delivery of Amphiphilic Polysiloxane Nanoparticles to Caveolae

Microdomains such as lipid raft and caveolae are organized as functional compartments in plasma membrane of cells. In this study, we note the functional platform of caveolae with dual functions, internalization of external substances and cell signalings leading to nitric oxide release, and hypothesize that the switching of enzyme activity of endothelial nitric oxide synthase can be achieved by targeting caveolae with nanoparticles. We prepared polysiloxane nanoparticles and studied cellular uptake of the nanoparticles and its concomitant influence on the nitric oxide release in human aortic endothelial cells. We found that polysiloxane nanoparticles were endocytosed via caveolae in human aortic endothelial cells and that enhanced nitric oxide release was followed by the cellular uptake of the nanoparticles. Furthermore, we confirmed that endothelial nitric oxide synthase was activated during cellular uptake of the nanoparticles. These findings support our idea that delivery of the polymeric nanoparticles to endothelial cells can lead to the induction of nitric oxide release.

Compartmentalizing VEGF-Induced ERK2/1 Signaling in Placental Artery Endothelial Cell Caveolae: A Paradoxical Role of Caveolin-1 in Placental Angiogenesis in Vitro

On vascular endothelial growth factor (VEGF) stimulation, both VEGF R1 and R2 receptors were phosphorylated in ovine fetoplacental artery endothelial (oFPAE) cells. Treatment with VEGF stimulated both time- and dose-dependent activation of ERK2/1 in oFPAE cells. VEGF-induced ERK2/1 activation was mediated by VEGFR2, but not VEGFR1, and was linked to intracellular calcium, protein kinase C, and Raf-1. VEGF stimulated oFPAE cell proliferation, migration, and tube formation in vitro. Blockade of ERK2/1 pathway attenuated VEGF-induced cell proliferation and tube formation but failed to inhibit migration in oFPAE cells. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin or by down-regulation of its structural protein caveolin-1 blunted VEGF-induced ERK2/1 activation, proliferation, and tube formation in oFPAE cells, indicating an essential role of integral caveolae in these VEGF-induced responses. Adenoviral overexpression of caveolin-1 and addition of a caveolin scaffolding domain peptide also inhibited VEGF-stimulated ERK2/1 activation, cell proliferation, and tube formation in oFPAE cells. Furthermore, molecules comprising the ERK2/1 signaling module, including VEGFR2, protein kinase Calpha, Raf-1, MAPK kinase 1/2, and ERK2/1, resided with caveolin-1 in caveolae. VEGF transiently stimulated ERK2/1 activation in the caveolae similarly as in intact cells. Caveolae disruption greatly diminished ERK2/1 activation by VEGF in oFPAE cell caveolae. We conclude that caveolae function as a platform for compartmentalizing the VEGF-induced ERK2/1 signaling module. Caveolin-1 and caveolae play a paradoxical role in regulating VEGF-induced ERK2/1 activation and in vitro angiogenesis as evidenced by the similar inhibitory effects of down-regulation and overexpression of caveolin-1 and disruption of caveolae in oFPAE cells.

Caveolae and neurotrophins in pulmonary artery smooth muscle

The mechanisms underlying pulmonary hypertension are still being explored. Recent studies suggest a role for caveolae (pit‐like plasma membrane structures), although it is not entirely clear whether they enhance or blunt pulmonary hypertension. The proteins caveolins ‐1, and ‐2, and polymerase I and transcription release factor (PTRF) are essential to forming caveolae. In other tissues, growth factors called neurotrophins (e.g. brain‐derived neurotrophic factor, BDNF) are thought to affect cell proliferation and structure. This study investigated caveolins and neurotrophins in human pulmonary artery. Western analysis of artery from a patient with primary pulmonary hypertension showed decreased levels of caveolin‐1, caveolin‐2, PTRF and RhoA. Exposure of human pulmonary artery to 1 nM BDNF increased protein levels of caveolin‐1 and PTRF. Isolated pulmonary artery smooth muscle cells exposed to BDNF showed decreased [Ca2+]i response to bradykinin (10 nM) and histamine (10 μM), but no change in caveolin or PTRF levels. These data suggest that decreased caveolin levels may contribute to increased pulmonary vascular contractility, while neurotrophins (potentially by increasing caveolin levels) may alleviate excessive constriction. Further studies are required to determine whether neurotrophins affect caveolae in endothelial cells or smooth muscle.Supported by FAMRI, NIH grant UL1RR024150, and the Department of Anesthesiology, Mayo Clinic.

Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation

Localization of proteases to the surface of endothelial cells and remodeling of the extracellular matrix (ECM) are essential to endothelial cell tube formation and angiogenesis. Here, we partially localized active cathepsin B and its cell surface binding partners, S100A/p11 (p11) of the annexin II heterotetramer (AIIt), to caveolae of human umbilical vein endothelial cells (HUVEC). Via a live-cell proteolysis assay, we observed that degradation products of quenched-fluorescent (DQ)-proteins (i.e. gelatin and collagen IV) colocalized intracellularly with caveolin-1 (cav-1) of HUVEC grown in either monolayer cultures or in vitro tube formation assays. Activity-based probes that bind covalently to active cysteine cathepsins and degradation products of DQ-collagen IV partially localized to intracellular vesicles that contained cav-1 and active cysteine cathepsins. Biochemical analyses revealed that the distribution of active cathepsin B in caveolar fractions increased during in vitro tube formation. Pro-uPA, uPAR, MMP-2 and MMP-14, which have been linked with cathepsin B to ECM degradation pathways, were also found to increase in caveolar fractions during in vitro tube formation. Our findings are the first to demonstrate through live-cell imaging ECM degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation.

Guanosine Triphosphate Cyclohydrolase I Expression and Enzymatic Activity Are Present in Caveolae of Endothelial Cells

Tetrahydrobiopterin is an essential cofactor required for the synthesis of NO. GTP cyclohydrolase I (GTPCH I) is the rate-limiting enzyme for tetrahydrobiopterin production in endothelial cells, yet little is known about the subcellular localization of this enzyme. In this study, we demonstrated that GTPCH I is localized to caveolar membrane microdomains along with caveolin-1 and endothelial NO synthase. GTPCH I activity was detected in isolated caveolar membranes from cultured endothelial cells. Confocal and electron microscopy analyses confirmed GTPCH I colocalization with caveolin-1. Consistent with in vitro studies, GTPCH I activity was evident in isolated caveolar microdomains from lung homogenates of wild-type mice. Importantly, a 2-fold increase in GTPCH I activity was detected in the aortas of caveolin-1-deficient mice, suggesting that caveolin-1 may be involved in the control of GTPCH I enzymatic activity. Indeed, overexpression of caveolin-1 inhibits GTPCH I activity, and tetrahydrobiopterin biosynthesis is activated by the disruption of caveolae structure. These studies demonstrate that GTPCH I is targeted to caveolae microdomains in vascular endothelial cells, and tetrahydrobiopterin production occurs in close proximity to endothelial NO synthase. In addition, our findings provide new insights into the regulation of GTPCH I activity by the caveolar coat protein, caveolin-1.

Endothelial Caveolin-1 Regulates Pathologic Angiogenesis in a Mouse Model of Colitis

Increased vascular density has been associated with progression of human inflammatory bowel diseases (IBDs) and animal models of colitis. Pathologic angiogenesis in chronically inflamed tissues is mediated by several factors that are regulated at specialized lipid rafts known as caveolae. Caveolin-1 (Cav-1), the major structural protein of caveolae in endothelial cells, is involved in the regulation of angiogenesis, so we investigated its role in experimental colitis. Colitis was induced by administration of dextran sodium sulfate to wild-type and Cav-1(-/-) mice, as well as Cav-1(-/-) mice that overexpress Cav-1 only in the endothelium. Colon tissues were analyzed by histologic analyses. Leukocyte recruitment was analyzed by intravital microscopy; angiogenesis was evaluated by immunohistochemistry and in vivo disk assays. Cav-1 protein levels increased after the induction of colitis in wild-type mice. In Cav-1(-/-) mice or mice given a Cav-1 inhibitory peptide, the colitis histopathology scores, vascular densities, and levels of inflammatory infiltrates decreased significantly compared with controls. Lower levels of leukocyte and platelet rolling and adhesion colitis also were observed in Cav-1(-/-) mice and mice given a Cav-1 inhibitory peptide, compared with controls. Cav-1(-/-) mice that received transplants of wild-type bone marrow had a lower colitis score than wild-type mice. Data from mice that overexpress Cav-1 only in the endothelium indicated that endothelial Cav-1 is the critical regulator of colitis. Genetic deletion or pharmacologic inhibition of endothelial Cav-1 also significantly decreased vascular densities and angiogenesis scores, compared with controls. Endothelial Cav-1 mediates angiogenesis in experimental colitis. Modulation of Cav-1 could provide a novel therapeutic target for IBD.

Caveolae and transcytosis in endothelial cells: role in atherosclerosis

The endothelium plays an important role in the regulation of molecular exchanges between the blood and peripheral tissues. The transport of molecules between tissues must be tightly controlled in order to maintain homeostasis between the different organs of the body. The endothelial transcytosis pathway has been shown to direct the transfer of proteins and solutes and therefore to act as a filtering system. This transport mode has been demonstrated to involve plasma-membrane vesicles that may be transferred with their cargo components from the apical to the basal side of endothelial cells. Among the vesicles implicated in the regulation of transcytosis, caveolae, which are 50 to 100-nm plasma-membrane invaginations, have been reported to play an essential part. In this paper, we review the function of caveolae and their major protein component (i.e., caveolin-1) in the regulation of endothelial transcytosis. The roles of caveolae in vascular diseases, such as atherosclerosis, are discussed.

Abstract 777: Caveolin-1 Regulates NADPH Oxidase Subcellular Location And Activity In Endothelial Cells.

Background: The biological setting or location where reactive oxygen species (ROS) are produced is a new concept which may provide a mechanistic explanation for redox signaling specificity. In this regard, we recently reported that NADPH oxidase, a major superoxide producing enzyme system, is localized and functional in endothelial cell caveolae. Here, we investigate the role of caveolin-1, the main structural protein of caveolae, in the organization and regulation of NADPH oxidase activity and ROS mediated responses to cytokine challenge. Methods and Results: Superoxide production was measured by DHE assay in endothelial cells derived from wild type (wt) and caveolin-1 knockout (Cav−/ −) mouse lungs (MLEC). TNFα failed to induced superoxide production in Cav−/ − endothelial cell cultures (MOI = 0). However, infection of knockout cells with full-length caveolin-1 containing Adenovirus at MOI of 10 and 50 progressively restored TNFα effect on superoxide production with MOI 50 nearly reaching those observed in wt cells. The distribution of NADPH oxidase within the plasma membrane showed localization to membrane rafts fractions in wt cells which shifted to the non-raft membrane fractions in Cav-1−/ − MLEC. Re-expression of caveolin-1 (MOI 50) restored raft targeting of NADPH oxidase and both gp91 and p22phox co-precipitated with newly expressed caveolin-1 immunoprecipitated from raft fractions. To evaluate the cellular consequences of a potential redox signaling relay involving caveolin-1, we examined an NADPH oxidase dependent pathway involving activation of JNK1/2 MAP kinases and downstream upregulation of ICAM. We found that phosphorylation of JNK1/2 observed in wt MLEC in response to TNFα was significantly attenuated in Cav−/ − MLEC. In addition, TNFα did not induce upregulation of ICAM expression in caveolin-1 knockout MLEC or wt cells pretreated with DPI. Conclusions: Collectively, these results indicate that caveolin-1 is a necessary component of the molecular machinery for proper subcellular positioning required for cytokine activation of NADPH oxidase and superoxide production in endothelial cells. Thus, caveolin-1 may constitute a potential therapeutic target for altering ROS associated with cardiovascular pathology.

Caveolin-associated Accumulation of Globotriaosylceramide in the Vascular Endothelium of α-Galactosidase A Null Mice

Cardiovascular complications, including stroke and myocardial infarction, result in premature mortality in patients with Fabry disease, an X-linked deficiency of alpha-galactosidase A (alpha-Gal A). The enzymatic defect results in the deposition of globotriaosylceramide (Gb3) in the vascular endothelium. To better understand the underlying pathogenesis of Fabry disease, the caveolar lipid content of primary cultured mouse aortic endothelial cells isolated from alpha-Gal A null mice was measured. Lipid mass analysis revealed that the excessive Gb3 in cultured alpha-Gal A-deficient mouse aortic endothelial cells accumulated in endothelial plasma membrane caveolar fractions. The levels of glucosylceramide and lactosylceramide increased in parallel with Gb3 levels in an age-dependent manner, whereas globotetraosylceramide (Gb4) levels reached maximal levels by 6 months of age and then rapidly decreased at older ages. The levels of cholesterol enriched in caveolar membranes declined in parallel with the progressive deposition of Gb3. Depleting Gb3 with recombinant human alpha-Gal A protein or d-threo-ethylenedioxyphenyl-P4, an inhibitor of glucosylceramide synthase, restored cholesterol in cultured alpha-Gal A-deficient mouse aortic endothelial cell caveolae. By contrast, recombinant human alpha-Gal A was less effective in normalizing the cholesterol content. These results demonstrate the caveolar accumulation of glycosphingolipids in an in vitro model of a lysosomal storage disease and raise the possibility that dynamic changes in the composition of plasma membrane lipid microdomains may mediate the endothelial dysfunction seen in Fabry disease.

Endothelial cytoskeletal reorganization in response to PAR1 stimulation is mediated by membrane rafts but not caveolae

The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its NH(2) terminus. PAR1 activation stimulates the RhoA/Rho kinase signaling cascade, leading to myosin light chain (MLC) phosphorylation, actin stress fiber formation, and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5 mug/ml, 5 min) to ablate lipid rafts before thrombin (100 nM) or PAR agonist stimulation. We found that diphosphorylation of MLC and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used a small interferring RNA approach to knockdown caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.