20-Hydroxyeicosatetraenoic Acid Impairs Endothelial Insulin Signaling by Inducing Phosphorylation of the Insulin Receptor Substrate-1 at Ser616

20-hydroxyeicosatetraenoic acid (20-HETE) induces endothelial dysfunction and is correlated with diabetes. This study was designed to investigate the effects of 20-HETE on endothelial insulin signaling.Human umbilical vein endothelial cells (HUVECs) or C57BL/6J mice were treated with 20-HETE in the presence or absence of insulin, and p-ERK1/2, p-JNK, IRS-1/PI3K/AKT/eNOS pathway, were examined in endothelial cells and aortas by immunoblotting. eNOS activity and nitric oxide production were measured. 20-HETE increased ERK1/2 phosphorylation and IRS-1 phosphorylation at Ser⁶¹⁶; these effects were reversed by ERK1/2 inhibition. We further observed that 20-HETE treatment resulted in impaired insulin-stimulated IRS-1 phosphorylation at Tyr⁶³² and subsequent PI3-kinase/Akt activation. Furthermore, 20-HETE treatment blocked insulin-stimulated phosphorylation of eNOS at the stimulatory Ser¹¹⁷⁷ site, eNOS activation and NO production; these effects were reversed by inhibiting ERK1/2. Treatment of C57BL/6J mice with 20-HETE resulted in ERK1/2 activation and impaired insulin-dependent activation of the IRS-1/PI3K/Akt/eNOS pathway in the aorta. Our data suggest that the 20-HETE activation of IRS-1 phosphorylation at Ser⁶¹⁶ is dependent on ERK1/2 and leads to impaired insulin-stimulated vasodilator effects that are mediated by the IRS-1/PI3K/AKT/eNOS pathway.     

Coordinated regulation of endothelial calcium signaling and shear stress-induced nitric oxide production by PKCβ and PKCη

BackgroundProtein Kinase C (PKC) is a promiscuous serine/threonine kinase regulating vasodilatory responses in vascular endothelial cells. Calcium-dependent PKCbeta (PKCβ) and calcium-independent PKCeta (PKCη) have both been implicated in the regulation and dysfunction of endothelial responses to shear stress and agonists.ObjectiveWe hypothesized that PKCβ and PKCη differentially modulate shear stress-induced nitric oxide (NO) production by regulating the transduced calcium signals and the resultant eNOS activation. As such, this study sought to characterize the contribution of PKCη and PKCβ in regulating calcium signaling and endothelial nitric oxide synthase (eNOS) activation after exposure of endothelial cells to ATP or shear stress.MethodsBovine aortic endothelial cells were stimulated in vitro under pharmacological inhibition of PKCβ with LY333531 or PKCη targeting with a pseudosubstrate inhibitor. The participation of PKC isozymes in calcium flux, eNOS phosphorylation and NO production was assessed following stimulation with ATP or shear stress.ResultsPKCη proved to be a robust regulator of agonist- and shear stress-induced eNOS activation, modulating calcium fluxes and tuning eNOS activity by multi-site phosphorylation. PKCβ showed modest influence in this pathway, promoting eNOS activation basally and in response to shear stress. Both PKC isozymes contributed to the constitutive and induced phosphorylation of eNOS. The observed PKC signaling architecture is intricate, recruiting Src to mediate a portion of PKCη's control on calcium entry and eNOS phosphorylation. Elucidation of the importance of PKCη in this pathway was tempered by evidence of a single stimulus producing concurrent phosphorylation at ser1179 and thr497 which are antagonistic to eNOS activity.ConclusionsWe have, for the first time, shown in a single species in vitro that shear stress- and ATP-stimulated NO production are differentially regulated by classical and novel PKCs. This study furthers our understanding of the PKC isozyme interplay that optimizes NO production. These considerations will inform the ongoing design of drugs for the treatment of PKC-sensitive cardiovascular pathologies.     

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.     

ICAM-1-coupled cytoskeletal rearrangements and transendothelial lymphocyte migration involve intracellular calcium signaling in brain endothelial cell lines

Endothelium of the cerebral blood vessels, which constitutes the blood-brain barrier, controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells using two rat brain endothelial cell lines (RBE4 and GP8), we report in this paper that ICAM-1 cross-linking induces a sustained tyrosine phosphorylation of the phosphatidylinositol-phospholipase C (PLC)gamma1, with a concomitant increase in both inositol phosphate production and intracellular calcium concentration. Our results suggest that PLC are responsible, via a calcium- and protein kinase C (PKC)-dependent pathway, for p60Src activation and tyrosine phosphorylation of the p60Src substrate, cortactin. PKCs are also required for tyrosine phosphorylation of the cytoskeleton-associated proteins, focal adhesion kinase and paxillin, but not for ICAM-1-coupled p130Cas phosphorylation. PKC's activation is also necessary for stress fiber formation induced by ICAM-1 cross-linking. Finally, cell pretreatment with intracellular calcium chelator or PKC inhibitors significantly diminishes transmonolayer migration of activated T lymphocytes, without affecting their adhesion to brain endothelial cells. In summary, our data demonstrate that ICAM-1 cross-linking induces calcium signaling which, via PKCs, mediates phosphorylation of actin-associated proteins and cytoskeletal rearrangement in brain endothelial cell lines. Our results also indicate that these calcium-mediated intracellular events are essential for lymphocyte migration through the blood-brain barrier.     

Endothelial cell cortactin coordinates intercellular adhesion molecule-1 clustering and actin cytoskeleton remodeling during polymorphonuclear leukocyte adhesion and transmigration

Endothelial cell ICAM-1 interacts with leukocyte beta(2) integrins to mediate adhesion and transmit outside-in signals that facilitate leukocyte transmigration. ICAM-1 redistribution and clustering appear necessary for leukocyte transmigration, but the mechanisms controlling ICAM-1 redistribution and clustering have not been identified. We recently reported that Src kinase phosphorylation of endothelial cortactin regulates polymorphonuclear cell (PMN) transmigration. In this study, we tested the hypotheses that the Src family kinase-cortactin pathway mediates association of ICAM-1 with the actin cytoskeleton and that this association is required for ICAM-1 clustering and leukocyte transmigration. Cross-linking ICAM-1 induced cytoskeletal remodeling and a decrease in ICAM-1 lateral mobility, as assessed by fluorescence recovery after photobleaching. Cytoskeletal remodeling after ICAM-1 cross-linking was reduced by knockdown of cortactin by small interfering RNA, by expression of a cortactin mutant deficient in Src phosphorylation sites (cortactin3F), and by the Src kinase inhibitor PP2. Pretreatment of cytokine-activated human endothelial monolayers with cortactin small interfering RNA significantly decreased both actin and ICAM-1 clustering around adherent PMN and the formation of actin-ICAM-1 clusters required for PMN transmigration. Our data suggest a model in which tyrosine phosphorylation of cortactin dynamically links ICAM-1 to the actin cytoskeleton, enabling ICAM-1 to form clusters and facilitate leukocyte transmigration.     

Role of G(i/o)-Src kinase-PI3K/Akt pathway and caveolin-1 in β₂-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G_i/o proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G_i/o proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser¹¹⁷⁷, which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr¹⁴, which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G_i/o-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser¹¹⁷⁷ leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr¹⁴, through a G_i/o-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.     

Cryptococcus neoformans Activates RhoGTPase Proteins Followed by Protein Kinase C,Focal Adhesion Kinase,and Ezrin to Promote Traversal across the Blood-Brain Barrier

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis. Previous studies have demonstrated that Cryptococcus binding and invasion of human brain microvascular endothelial cells (HBMEC) is a prerequisite for transmigration across the blood-brain barrier. However, the molecular mechanism involved in the cryptococcal blood-brain barrier traversal is poorly understood. In this study we examined the signaling events in HBMEC during interaction with C. neoformans. Analysis with inhibitors revealed that cryptococcal association, invasion, and transmigration require host actin cytoskeleton rearrangement. Rho pulldown assays revealed that Cryptococcus induces activation of three members of RhoGTPases, e.g. RhoA, Rac1, and Cdc42, and their activations are required for cryptococcal transmigration across the HBMEC monolayer. Western blot analysis showed that Cryptococcus also induces phosphorylation of focal adhesion kinase (FAK), ezrin, and protein kinase C α (PKCα), all of which are involved in the rearrangement of host actin cytoskeleton. Down-regulation of FAK, ezrin, or PKCα by shRNA knockdown, dominant-negative transfection, or inhibitors significantly reduces cryptococcal ability to traverse the HBMEC monolayer, indicating their positive role in cryptococcal transmigration. In addition, activation of RhoGTPases is the upstream event for phosphorylation of FAK, ezrin, and PKCα during C. neoformans-HBMEC interaction. Taken together, our findings demonstrate that C. neoformans activates RhoGTPases and subsequently FAK, ezrin, and PKCα to promote their traversal across the HBMEC monolayer, which is the critical step for cryptococcal brain infection and development of meningitis.     

Signaling pathways involving the sodium pump stimulate NO production in endothelial cells

The cardiac steroid ouabain, a known inhibitor of the sodium pump (Na⁺,K⁺-ATPase), has been shown to release endothelin from endothelial cells when used at concentrations below those that inhibit the pump. The present study addresses the question of which signaling pathways are activated by ouabain in endothelial cells. Our findings indicate that ouabain, applied at low concentrations to human umbilical cord endothelial cells (HUAECs), induces a reaction cascade that leads to translocation of endothelial nitric oxide synthase (eNOS) and to activation of phosphatidylinositol 3-kinase (PI3K). These events are followed by phosphorylation of Akt (also known as protein kinase B, or PKB) and activation of eNOS by phosphorylation. This signaling pathway, which results in increased nitric oxide (NO) production in HUAECs, is inhibited by the PI3K-specific inhibitor LY294002. Activation of the reaction cascade is not due to endothelin-1 (ET-1) binding to the ET-1 receptor B (ET_B), since application of the ET_B-specific antagonist BQ-788 did not have any effect on Akt or eNOS phosphorylation. The results shown here indicate that ouabain binding to the sodium pump results in the activation of the proliferation and survival pathways involving PI3K, Akt activation, stimulation of eNOS, and production of NO in HUAECs. Together with results from previous publications, the current investigation implies that the sodium pump is involved in vascular tone regulation.     

Forskolin increases angiogenesis through the coordinated cross-talk of PKA-dependent VEGF expression and Epac-mediated PI3K/Akt/eNOS signaling

Forskolin, a potent activator of adenylyl cyclases, has been implicated in modulating angiogenesis, but the underlying mechanism has not been clearly elucidated. We investigated the signal mechanism by which forskolin regulates angiogenesis. Forskolin stimulated angiogenesis of human endothelial cells and in vivo neovascularization, which was accompanied by phosphorylation of CREB, ERK, Akt, and endothelial nitric oxide synthase (eNOS) as well as NO production and VEGF expression. Forskolin-induced CREB phosphorylation, VEGF promoter activity, and VEGF expression were blocked by the PKA inhibitor PKI. Moreover, phosphorylation of ERK by forskolin was inhibited by the MEK inhibitor PD98059, but not PKI. The forskolin-induced Akt/eNOS/NO pathway was completely inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002, but not significantly suppressed by PKI. These inhibitors and a NOS inhibitor partially inhibited forskolin-induced angiogenesis. The exchange protein directly activated by cAMP (Epac) activator, 8CPT-2Me-cAMP, promoted the Akt/eNOS/NO pathway and ERK phosphorylation, but did not induce CREB phosphorylation and VEGF expression. The angiogenic effect of the Epac activator was diminished by the inhibition of PI3K and MEK, but not by the PKA inhibitor. Small interfering RNA-mediated knockdown of Epac1 suppressed forskolin-induced angiogenesis and phosphorylation of ERK, Akt, and eNOS, but not CREB phosphorylation and VEGF expression. These results suggest that forskolin stimulates angiogenesis through coordinated cross-talk between two distinct pathways, PKA-dependent VEGF expression and Epac-dependent ERK activation and PI3K/Akt/eNOS/NO signaling.     

VCAM-1 activation of endothelial cell protein tyrosine phosphatase 1B

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)alpha activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCalpha in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCalpha and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.     

ADP Signaling in Vascular Endothelial Cells: ADP-DEPENDENT ACTIVATION OF THE ENDOTHELIAL ISOFORM OF NITRIC-OXIDE SYNTHASE REQUIRES THE EXPRESSION BUT NOT THE KINASE ACTIVITY OF AMP-ACTIVATED PROTEIN KINASE*

ADP responses underlie therapeutic approaches to many cardiovascular diseases, and ADP receptor antagonists are in widespread clinical use. The role of ADP in platelet biology has been extensively studied, yet ADP signaling pathways in endothelial cells remain incompletely understood. We found that ADP promoted phosphorylation of the endothelial isoform of nitric-oxide synthase (eNOS) at Ser¹¹⁷⁹ and Ser⁶³⁵ and dephosphorylation at Ser¹¹⁶ in cultured endothelial cells. Although eNOS activity was stimulated by both ADP and ATP, only ADP signaling was significantly inhibited by the P2Y₁ receptor antagonist MRS 2179 or by knockdown of P2Y₁ using small interfering RNA (siRNA). ADP activated the small GTPase Rac1 and promoted endothelial cell migration. siRNA-mediated knockdown of Rac1 blocked ADP-dependent eNOS Ser¹¹⁷⁹ and Ser⁶³⁵ phosphorylation, as well as eNOS activation. We analyzed pathways known to regulate eNOS, including phosphoinositide 3-kinase/Akt, ERK1/2, Src, and calcium/calmodulin-dependent kinase kinase-β (CaMKKβ) using the inhibitors wortmannin, PD98059, PP2, and STO-609, respectively. None of these inhibitors altered ADP-modulated eNOS phosphorylation. In contrast, siRNA-mediated knockdown of AMP-activated protein kinase (AMPK) inhibited ADP-dependent eNOS Ser⁶³⁵ phosphorylation and eNOS activity but did not affect eNOS Ser¹¹⁷⁹ phosphorylation. Importantly, the AMPK enzyme inhibitor compound C had no effect on ADP-stimulated eNOS activity, despite completely blocking AMPK activity. CaMKKβ knockdown suppressed ADP-stimulated eNOS activity, yet inhibition of CaMKKβ kinase activity using STO-609 failed to affect eNOS activation by ADP. These data suggest that the expression, but not the kinase activity, of AMPK and CaMKKβ is necessary for ADP signaling to eNOS.     

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling.     

Regulation of endothelial glutathione by ICAM-1 governs VEGF-A-mediated eNOS activity and angiogenesis

Previous studies suggest that inflammatory cell adhesion molecules may modulate endothelial cell migration and angiogenesis through unknown mechanisms. Using a combination of in vitro and in vivo approaches, herein we reveal a novel redox-sensitive mechanism by which ICAM-1 modulates endothelial GSH that controls VEGF-A-induced eNOS activity, endothelial chemotaxis, and angiogenesis. In vivo disk angiogenesis assays showed attenuated VEGF-A-mediated angiogenesis in ICAM-1(-/-) mice. Moreover, VEGF-A-dependent chemotaxis, eNOS phosphorylation, and nitric oxide production were impaired in ICAM-1(-/-) mouse aortic endothelial cells (MAEC) compared to WT MAEC. Decreasing intracellular GSH in ICAM-1(-/-) MAEC to levels observed in WT MAEC with 150 microM buthionine sulfoximine restored VEGF-A responses. Conversely, GSH supplementation of WT MAEC with 5 mM glutathione ethyl ester mimicked defects observed in ICAM-1(-/-) cells. Deficient angiogenic responses in ICAM-1(-/-) cells were associated with increased expression of the lipid phosphatase PTEN, consistent with antagonism of signaling pathways leading to eNOS activation. PTEN expression was also sensitive to GSH status, decreasing or increasing in proportion to intracellular GSH concentrations. These data suggest a novel role for ICAM-1 in modulating VEGF-A-induced angiogenesis and eNOS activity through regulation of PTEN expression via modulation of intracellular GSH status.     

Analysis of the roles of ICAM-1 in neutrophil transmigration using a reconstituted mammalian cell expression model: implication of ICAM-1 cytoplasmic domain and Rho-dependent signaling pathway

Interaction between ICAM-1 (CD54) and fibrinogen (fg) has been shown to enhance leukocyte adhesion, but its specific role in the process of migration across endothelial cell junctions remains unclear. To overcome the problem of multiple adhesion receptors found on endothelial cells, we have engineered stable Chinese hamster ovary cell lines expressing ICAM-1 (Chinese hamster ovary ICAM-1). The transfection of ICAM-1 alone in these cells is sufficient to recapitulate the entire process of neutrophil adhesion and transmigration. This phenomenon was mediated by fg-ICAM-1 interactions, as depletion of fg, as well as the use of an Ab that specifically inhibits ICAM-1-fg interaction (2D5), completely abolished the effect of ICAM-1 expression on PMN transmigration. In addition, this ICAM-1-mediated transmigration is clearly dependent on the occurrence of fg-ICAM-1 interactions on the monolayer, and not on neutrophils, as the preincubation of the PMN with the mAb was ineffective. Furthermore, PMN transmigration, but not adhesion, is totally abolished when the ICAM-1 cytoplasmic domain is deleted, indicating that signaling inside the cell is required to mediate the fg-ICAM-1 effect on transmigration. Using a specific inhibitor of the small GTP-binding protein Rho, we have obtained evidence that this signaling cascade is involved. Thus, our results clearly show that ICAM-1 plays a key role in the migration of leukocytes across cell junctions, and indicate that this phenomenon is not a direct consequence of the enhanced adhesion mediated by the expression of ICAM-1.     

Cryptotanshinone inhibits oxidized LDL-induced adhesion molecule expression via ROS dependent NF-κB pathways

Adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, play important roles in the initial stage of atherosclerosis. Cryptotanshinone (CPT), a natural compound isolated from Salvia miltiorrhiza Bunge, exhibits anti-atherosclerotic activity although the underlying mechanisms remain elusive. In this study, the protective effect of CPT against oxidized low-density lipoprotein (ox-LDL)-induced adhesion molecule expression was investigated in human umbilical vein endothelial cells. Ox-LDL significantly induced ICAM-1, VCAM-1, and E-selectin expression at the mRNA and protein levels but reduced eNOS phosphorylation and NO generation, which were reversed by CPT pretreatment. Sodium nitroprusside, a NO donor, N-acetyl-L-cysteine (NAC), a reactive oxygen species (ROS) scavenger, and BAY117082, a NF-κB inhibitor, inhibited ox-LDL-induced ICAM-1, VCAM-1, and E-selectin expression. Ox-LDL-induced ROS production was significantly inhibited by CPT and NAC. Furthermore, ox-LDL activated the NF-κB signaling pathway by inducing phosphorylation of IKKβ and IκBα, promoting the interaction of IKKβ and IκBα, and increasing p65 nuclear translocation, which were significantly inhibited by CPT. In addition, CPT, NAC, and BAY117082 inhibited ox-LDL-induced membrane expression of ICAM-1, VCAM-1, E-selectin, and endothelial–monocyte adhesion and restored eNOS phosphorylation and NO generation. Results suggested that CPT inhibited ox-LDL-induced adhesion molecule expression by decreasing ROS and inhibiting the NF-κB pathways, which provides new insight into the anti-atherosclerotic mechanism of CPT.     

Epinephrine regulation of the endothelial nitric-oxide synthase: roles of RAC1 and beta3-adrenergic receptors in endothelial NO signaling

beta-Adrenergic receptors (betaAR) play an important role in vasodilation, but the mechanisms whereby adrenergic pathways regulate the endothelial isoform of nitric-oxide synthase (eNOS) are incompletely understood. We found that epinephrine significantly increases eNOS activity in cultured bovine aortic endothelial cells (BAEC). Epinephrine-dependent eNOS activation was accompanied by an increase in phosphorylation of eNOS at Ser(1179) and with decreased eNOS phosphorylation at the inhibitory phosphoresidues Ser(116) and Thr(497). Epinephrine promoted activation of the small G protein Rac1 and also led to the activation of protein kinase A. All of these responses to epinephrine in BAEC were blocked by the beta(3)AR blocker SR59230A. We transfected and validated duplex small interfering RNA (siRNA) constructs to selectively "knock down" specific signaling proteins in BAEC. siRNA-mediated knockdown of Rac1 completely blocked all beta(3)AR signaling to eNOS and also abrogated epinephrine-dependent cAMP-dependent protein kinase (PKA) and Akt activation. However, siRNA-mediated knockdown of PKA did not affect Rac1 activation by epinephrine but did attenuate Akt activation by epinephrine. These findings indicate that Rac1 is an upstream regulator of beta(3)AR signaling to PKA and to eNOS and identify a novel beta(3)AR --> Rac1 --> PKA --> Akt pathway in endothelium. We exploited the p21-activated kinase pulldown assay to identify proteins associated with activated Rac1 and found that epinephrine stimulated the association of eNOS with Rac1; epinephrine-stimulated eNOS-Rac1 interactions were blocked by the beta(3)AR antagonist SR59230A. Co-transfection of eNOS cDNA with constitutively active Rac1 enhanced beta(3)AR-promoted eNOS-Rac1 association; co-transfection of eNOS with dominant negative Rac1 completely blocked the eNOS-Rac1 association. We also found that epinephrine-induced Rac1 --> PKA --> Akt pathway mediates beta(3)AR-mediated endothelial cell migration. Taken together, our data establish that the small G protein Rac1 is a key regulator of beta(3)AR signaling in cultured aortic endothelial cells with potentially important implications for the pathways involved in adrenergic modulation of eNOS pathways in the vascular wall.     

eNOS activation mediated by AMPK after stimulation of endothelial cells with histamine or thrombin is dependent on LKB1

Reports on the role of AMP-activated protein kinase (AMPK) in thrombin-mediated activation of endothelial nitric-oxide synthase (eNOS) in endothelial cells have been conflicting. Previously, we have shown that under culture conditions that allow reduction of ATP-levels after stimulation, activation of AMPK contributes to eNOS phosphorylation and activation in endothelial cells after treatment with thrombin. In this paper we examined the signaling pathways mediating phosphorylation and activation of eNOS after stimulation of cultured human umbilical vein endothelial cells (HUVEC) with histamine and the role of LKB1-AMPK in the signaling. In Morgan's medium 199 intracellular ATP was lowered by treatment with histamine or the ionophore A23187 while in medium RMPI 1640 ATP was unchanged after identical treatment. In medium 199 inhibition of Ca^+ 2/CaM kinase kinase (CaMKK) by STO-609 only partially inhibited AMPK phosphorylation but after gene silencing of LKB1 with siRNA there was a total inhibition of AMPK phosphorylation by STO-609 after treatment with either histamine or thrombin, demonstrating phosphorylation of AMPK by both upstream kinases, LKB1 and CaMKK. Downregulation of AMPK with siRNA partially inhibited eNOS phosphorylation caused by histamine in cells maintained in medium 199. Downregulation of LKB1 by siRNA inhibited both phosphorylation and activity of eNOS and addition of the AMPK inhibitor Compound C had no further effect on eNOS phosphorylation. When experiments were carried out in medium 1640, STO-609 totally prevented the phosphorylation of AMPK without affecting eNOS phosphorylation. AMPKα2 downregulation resulted in a loss of the integrity of the endothelial monolayer and increased expression of GRP78, indicative of endoplasmic reticular (ER) stress. Downregulation of AMPKα1 had no such effect. The results show that culture conditions affect endothelial signal transduction pathways after histamine stimulation. Under conditions where intracellular ATP is lowered by histamine, AMPK is activated by both LKB1 and CaMKK and, in turn, mediates eNOS phosphorylation in an LKB1 dependent manner. Both AMPKα1 and − α2 are involved in the signaling. Under conditions where intracellular ATP is unchanged after histamine treatment, CaMKK alone activates AMPK and eNOS is phosphorylated and activated independent of AMPK.     

Regulation of Active ICAM-4 on Normal and Sickle Cell Disease RBCs via AKAPs Is Revealed by AFM

Human healthy (wild-type (WT)) and homozygous sickle (SS) red blood cells (RBCs) express a large number of surface receptors that mediate cell adhesion between RBCs, and between RBCs and white blood cells, platelets, and the endothelium. In sickle cell disease (SCD), abnormal adhesion of RBCs to endothelial cells is mediated by the intercellular adhesion molecule-4 (ICAM-4), which appears on the RBC membrane and binds to the endothelial αvβ3 integrin. This is a key factor in the initiation of vaso-occlusive episodes, the hallmark of SCD. A better understanding of the mechanisms that control RBC adhesion to endothelium may lead to novel approaches to both prevention and treatment of vaso-occlusive episodes in SCD. One important mechanism of ICAM-4 activation occurs via the cyclic adenosine monophosphate-protein kinase A (cAMP-PKA)-dependent signaling pathway. Here, we employed an in vitro technique called single-molecule force spectroscopy to study the effect of modulation of the cAMP-PKA-dependent pathway on ICAM-4 receptor activation. We quantified the frequency of active ICAM-4 receptors on WT-RBC and SS-RBC membranes, as well as the median unbinding force between ICAM-4 and αvβ3. We showed that the collective frequency of unbinding events in WT-RBCs is not significantly different from that of SS-RBCs. This result was confirmed by confocal microscopy experiments. In addition, we showed that incubation of normal RBCs and SS-RBCs with epinephrine, a catecholamine that binds to the β-adrenergic receptor and activates the cAMP-PKA-dependent pathway, caused a significant increase in the frequency of active ICAM-4 receptors in both normal RBCs and SS-RBCs. However, the unbinding force between ICAM-4 and the corresponding ligand αvβ3 remained the same. Furthermore, we demonstrated that forskolin, an adenylyl cyclase activator, significantly increased the frequency of ICAM-4 receptors in WT-RBCs and SS-RBCs, confirming that the activation of ICAM-4 is regulated by the cAMP-PKA pathway. Finally, we showed that A-kinase anchoring proteins play an essential role in ICAM-4 activation.