Basal and angiopoietin-1–mediated endothelial permeability is regulated by sphingosine kinase-1

Abstract

Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-1 (Ang-1)-regulated control of EC permeability is mediated by 2 different functional states of sphingosine kinase-1 (SK-1). Mice depleted of SK-1 have increased vascular leakiness, whereas mice transgenic for SK-1 in ECs show attenuation of leakiness. Furthermore, Ang-1 rapidly and transiently stimulates SK-1 activity and phosphorylation, and induces an increase in intracellular sphingosine-1-phosphate (S1P) concentration. Overexpression of SK-1 resulted in inhibition of permeability similar to that seen for Ang-1, whereas knockdown of SK-1 by small interfering RNA blocked Ang-1-mediated inhibition of permeability. Transfection with SK S225A , a nonphosphorylatable mutant of SK-1, inhibited basal leakiness, and both SK S225A and a dominant-negative SK-1 mutant removed the capacity of Ang-1 to inhibit permeability. These effects were independent of extracellular S1P as knockdown or inhibition of S1P 1 , S1P 2 , or S1P 3 , did not affect the Ang-1 response. Thus, SK-1 levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-1-induced inhibition of leakiness is mediated through activation of SK-1, defining a new signaling pathway in the Ang-1 regulation of permeability. IntroductionThe control of vascular homeostasis is mediated through the maintenance of endothelial cell (EC) monolayer integrity, which is responsible for the tonic impermeable nature of blood vessels. Altered endothelial integrity underlies changes in vascular permeability, as seen in inflammatory responses and angiogenesis, and in diseases, such as rheumatoid arthritis and atherosclerosis. Stimuli, such as thrombin, histamine, and tumor necrosis factor (TNF), are powerful inducers of EC permeability, 1 whereas the angiogenic factor angiopoietin-1 (Ang-1), acting through the tyrosine kinase receptor Tie2, and sphingosine-1-phosphate (S1P), acting through the G protein coupled receptors S1P 1 or S1P 3 , inhibit the action of these stimulators and are potent protectors of barrier function. [2][3][4] The Ang-1/Tie2 axis is essential for vascular development and, in the adult, for the formation and maintenance of EC integrity. 2 In vivo, transgenic expression of Ang-1 in mice results in leakage resistant blood vessels, 5 and systemic administration of Ang-1 inhibits vessel leak. 6 In vitro, the antipermeability and antiinflammatory effects of Ang-1 appear to function at 2 levels, one to inhibit the basal permeability of EC and the other to limit the activity of agents, such as thrombin and TNF. 3 The signaling pathways regulated in response to ligand-induced phosphorylation of Tie2 are only now being elucidated. 7 AKT is involved in the prosurvival and angiogenic activities of Ang-1. 8 Interaction of ShcA with Tie2 and phospholipase D-dependent regulation of mitogen-activated protein kinase (MAPK) are involved in Ang-1-mediated cell migration 9 and a Ras/MAPK cascade is responsible for Ang-1-induced ce...

S1P Is Associated with Protection in Human and Experimental Cerebral Malaria

“…Prophylactic treatment resulted in increased Ang1 levels, a biomarker of endothelial quiescence, which may explain the enhanced survival provided by prophylactic FTY720 treatment since in humans, decreased Ang1 levels are associated with CM (46). Increased Ang1 and S1P levels have also been associated with the maintenance of vascular integrity (47,48). S1P has been shown recently to induce nitric oxide (NO) (49) as well as vascular endothelial growth factor (VEGF) expression (50) in endothelial cells.…”

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“…Adapted from methods previously described, 29 Ang-2 siRNA (50 nmol/L) (HSS100480: 5Ј-AAUACUUCAGCA-CAGUCUCUGAAGC-3Ј;HSS100482:5Ј-UUCUCCUGAA-GGGUUACCAAAUCCC-3Ј) were transfected into HUVECs using HiPerfect (Qiagen, Doncaster, Victoria, Aust.) in EGMII medium (Clonetics/Lonza, Basel, Switzerland) when cells were at 60% confluency.…”

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“…24 SK-1 also has an agonist-induced activation state, which occurs, at least in response to tumor necrosis factor-␣ and phorbol esters, as a direct consequence of phosphorylation at serine 225 by extracellular signal-regulated kinase 1/2. 22 The effects of this single phosphorylation are twofold: it is required for agonist-induced increases in the catalytic activity of SK-1 and is necessary for translocation of this protein from the cytosol to the plasma membrane.…”

“…S1P also acts intracellularly and independent of S1P receptors to enhance EC proliferation and suppress apoptosis. 44 Increasing evidence for S1P as a bone fide second messenger 11 includes our own studies showing that S1P receptor inhibitors VPC23019 and JTE-013, although efficient for inhibiting agonist-induced SK-1 activity, 24,45 failed to have any effect on survival in the SK-1-overexpressing EC 24 or on the differentiation of endothelial progenitor cells. 46 Paik et al 47 previously described S1P 1 -and S1P 3 -mediated Rho signaling in ␣ v ␤ 3 activation for EC migration.…”

“…As adapted from methods described previously, 24 small interfering RNA (siRNA) targeted to human ␤ 3 (5Ј-CCA-CUGUAUAAAGAGGCCACGUCUA-3Ј and 5Ј-UAGACG-UGGCCUCUUUAUACAGUGG-3Ј) and control "Validated Stealth" siRNA were synthesized by Invitrogen before transfection into HUVECs using HiPerfect (Qiagen, Gaithersburg, MD) in EGMII medium (Clonetics, Lonza, Basel, Switzerland) when cells were at 60% confluent. After 24 hours, culture media were changed to complete Medium 199-20% fetal calf serum until cells were harvested at 48 hours post-transfection.…”