VE-cadherin and PECAM-1 enhance ALL migration across brain microvascular endothelial cell monolayers.

Objective: Infiltration of the central nervous system (CNS) by leukemia is a problematic disease manifestation of acute lymphoblastic leukemia (ALL). The mechanisms by which leukocytes interact with human brain-derived microvasculature endothelial cells (HBMEC) and enter the CNS are largely derived from models of inflammation. However, our data indicate that ALL cells do not elicit an inflammatory phenotype by HBMEC.

N-cadherin levels in endothelial cells are regulated by monolayer maturity and p120 availability.

Endothelial cells (ECs) express VE-cadherin and N-cadherin, and recent data suggest that VE-cadherin levels are dependent on N-cadherin expression. While investigating changes in N-cadherin levels during endothelial monolayer maturation, the authors found that VE-cadherin levels are maintained in ECs despite a decrease in N-cadherin, suggesting that VE-cadherin levels may not depend on N-cadherin. Knockdown of N-cadherin did not affect VE-cadherin levels in ECs with low endogenous N-cadherin expression.

Sphingosine 1-phosphate rapidly increases endothelial barrier function independently of VE-cadherin but requires cell spreading and Rho kinase.

Sphingosine 1-phosphate (S1P) rapidly increases endothelial barrier function and induces the assembly of the adherens junction proteins vascular endothelial (VE)-cadherin and catenins. Since VE-cadherin contributes to the stabilization of the endothelial barrier, we determined whether the rapid, barrier-enhancing activity of S1P requires VE-cadherin. Ca(2+)-dependent, homophilic VE-cadherin binding of endothelial cells, derived from human umbilical veins and grown as monolayers, was disrupted with EGTA, an antibody to the extracellular domain of VE-cadherin, or gene silencing of VE-cadherin with small interfering RNA.

Membrane fusion by VAMP3 and plasma membrane t-SNAREs.

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration.

Sphingosine 1-phosphate prevents platelet-activating factor-induced increase in hydraulic conductivity in rat mesenteric venules: pertussis toxin sensitive.

Sphingosine 1-phosphate (S1P) is a biologically active lipid. In vitro, S1P tightens the endothelial barrier, as assessed by a rapid increase in electrical resistance and a decrease in solute permeability. We hypothesized that this activity of S1P would also occur in vivo.

RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability.

Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses.

VE-cadherin-p120 interaction is required for maintenance of endothelial barrier function.

Interaction of p120 with juxtamembrane domain (JMD) of VE-cadherin has been implicated in regulation of endothelial cell-cell adhesion. We used a number of approaches to alter the level of p120 available for binding to VE-cadherin as a means to investigate the role of p120-VE-cadherin interaction in regulation of barrier function in confluent endothelial monolayers. Expression of an epitope-tagged fragment corresponding to JMD of VE-cadherin resulted in a decrease in endothelial barrier function as assessed by changes in albumin clearance and electrical resistance.

p38 MAPK activation by TGF-beta1 increases MLC phosphorylation and endothelial monolayer permeability.

Transforming growth factor (TGF)-beta1 increases endothelial monolayer permeability and myosin light chain phosphorylation (MLC-P) beginning 1-2 h posttreatment, suggesting that changes in gene expression may be required for these responses. The role of extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinase (p38 MAPK) was investigated because both kinases have been implicated in regulating gene expression after TGF-beta1. ERK1/2 phosphorylation increased threefold above the control level, and the increase was temporally associated with the increase in MLC-P.

Platelet lipid(s) bound to albumin increases endothelial electrical resistance: mimicked by LPA.

The objectives were to determine whether the permeability-decreasing activity of platelet-conditioned medium (PCM) is associated with a lipid bound to albumin and whether lysophosphatidic acid (LPA) is present in the PCM. A decrease in permeability was assessed by an increase in electrical resistance across endothelial cell monolayers derived from bovine pulmonary arteries and microvessels. The Sephacryl S-200 fraction of PCM that contained albumin, the albumin immunoprecipitate from the PCM, and the methanol extract from the albumin immunoprecipitate all increased endothelial electrical resistance.

Platelet-conditioned medium increases endothelial electrical resistance independently of cAMP/PKA and cGMP/PKG.

Platelets release a soluble factor into blood and conditioned medium (PCM) that decreases vascular endothelial permeability. The objective of this study was to determine the signal-transduction pathway that elicits this decrease in permeability. Permeability-decreasing activity of PCM was assessed by the real-time measurement of electrical resistance across cell monolayers derived from bovine pulmonary arteries and microvessels.

Electrical impedance of cultured endothelium under fluid flow.

The morphological and functional status of organs, tissues, and cells can be assessed by evaluating their electrical impedance. Fluid shear stress regulates the morphology and function of endothelial cells in vitro. In this study, an electrical biosensor was used to investigate the dynamics of flow-induced alterations in endothelial cell morphology in vitro.

Statin drugs and dietary isoprenoids as antithrombotic agents.

Statin drugs and various isoprenoids from plant origins inhibit mevalonic acids, cholesterol, and other isoprenoid products. Among these, reduction of farnesyl and geranylgeranyl prenylated proteins impedes signal transduction at the cellular level. The authors envision that limiting such prenylated proteins downregulates thrombin-stimulated events, including decreasing the expression and availability of protease-activated receptor-1 mitigating thrombin stimulation of cells, tissue factor preventing additional thrombin generation, and plasminogen activator inhibitor-1 allowing thrombosis.

Oxidant-increased endothelial permeability: prevention with phosphodiesterase inhibition vs. cAMP production.

The present objective was to determine whether hydrogen peroxide (H(2)O(2)) increases transvascular albumin clearance and lung weight in an isolated rat lung and whether posttreatment with cAMP-enhancing agents can prevent these increases. Transvascular albumin clearance was assessed by (125)I-labeled albumin clearance ((125)I-albumin flux/perfusate concentration of (125)I-albumin) at a given fluid filtration. Nonlinear regression analysis of transvascular albumin clearance vs.

Increased recycling of (alpha)5(beta)1 integrins by lung endothelial cells in response to tumor necrosis factor.

Tumor necrosis factor (alpha) (TNF-(alpha) can change the interaction of lung endothelial cell monolayers with their extracellular matrix in association with an increase in endothelial monolayer protein permeability. Using immunofluorescence microscopy and flow cytometry, we determined if exposure of calf pulmonary artery endothelial monolayers to TNF-(alpha) may influence cell-matrix interactions by altering the clustering as well as internalization of the (&agr;)5(beta)1 integrins (or fibronectin receptors) on the surface of endothelial cells. Immunofluorescence microscopy revealed that TNF-(alpha) caused an increase in the intracellular staining of (alpha)5(alpha)1 integrins within structures similar to endocytic vesicles as well as an increase in antibody-induced clustering of the integrins at the cell periphery.

Rearrangement of adherens junctions by transforming growth factor-beta1: role of contraction.

The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-beta1 (TGF-beta1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-beta1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-beta1 treatment and continued to slowly decrease over the next 6 h.

Protein, not adenosine or adenine nucleotides, mediates platelet decrease in endothelial permeability.

Platelets and platelet-conditioned medium (PCM) decrease endothelial protein permeability in vitro. Adenosine and a > 100-kDa protein have previously been implicated as the soluble factors released from platelets that decrease endothelial permeability. The objective of this study was to further investigate the role of adenosine in this platelet response.

Contrasting effects of hypochlorous acid and hydrogen peroxide on endothelial permeability: prevention with cAMP drugs.

Activated polymorphonuclear leukocytes generate a cascade of reduced oxygen metabolites. In addition to their antimicrobial role, hydrogen peroxide (H2O2) and hypochlorous acid (HOCl) function as inflammatory mediators and increase the protein permeability of the vascular endothelium. The objectives of the present study were to compare the effects of H2O2 and HOCl with respect to relative potencies and the time course and magnitude of changes in cell shape and permeability of endothelial cell monolayers derived from bovine pulmonary artery, to determine if HOCl produced by conversion of H2O2 with myeloperoxidase and Cl- produces comparable results as the direct administration of HOCl, and to show that adenosine 3',5'-cyclic monophosphate (cAMP)-enhancing agents can prevent the increased endothelial permeability induced by HOCl and H2O2.

The cigarette tar component p-benzoquinone blocks T-lymphocyte activation by inhibiting interleukin-2 production, but not CD25, ICAM-1, or LFA-1 expression.

Cigarette smoking has been shown to cause profound suppression of T-cell responses in the lungs, but the mechanism by which this phenomenon occurs is not known. We have shown that 10 microM p-benzoquinone (p-BQ), a thiol-reactive benzene derivative found in cigarette tar, inhibits mitogen-induced IL-2 production by human peripheral blood mononuclear cells by 76 +/- 7% without affecting lymphocyte/macrophage agglutination or blast transformation. The effect of p-BQ appeared to be specific for IL-2 production, since de novo induction of the IL-2 receptor alpha-chain (CD25) and ICAM-1 (CD54) and upregulation of LFA-1 alpha/beta (CD11a and CD18) were unaffected.

Thrombin increases fluid flux in isolated rat lungs by a hemodynamic and not a permeability mechanism.

Alpha-Thrombin increases endothelial protein permeability in vitro and induces weight gain in the isolated perfused lung. The objectives of this study were to determine whether thrombin increases endothelial permeability of the isolated perfused rat lung and whether a change in permeability or hemodynamics mediates the gain in lung weight. Endothelial protein permeability was assessed by regression analysis of 125I-labeled albumin clearance vs.

Fibronectin inhibition of platelet thrombus formation in an in vivo porcine model of vascular injury.

Platelets adhere and aggregate in response to exposed subendothelial matrix during vascular injury. The present study examines the effect of plasma fibronectin on platelet deposition at a site of vascular injury in an in vivo porcine model. The internal carotid arteries in anesthetized Yorkshire pigs were bilaterally exposed and the distal half of each vessel stripped of endothelium.

Isoproterenol decreases protein permeability in edematous isolated rabbit lungs: estimation of PS and sigma.

The objective of the present study was to determine whether the ability of the beta-adrenergic agonist isoproterenol to attenuate pulmonary edema occurs via a permeability and/or hemodynamic mechanism. In isolated perfused rabbit lungs, the restrictive property of the vascular barrier to the movement of fluid and protein was assessed by measurements of the capillary filtration coefficient (Kf) and the transvascular clearance of 125I-labeled albumin, respectively. Regression analysis of albumin clearance vs.

Delayed elevation of ED1-cellular fibronectin in plasma following postsurgical bacteremia.

Fibronectin (Fn) exists in both a soluble form in plasma and lymph as well as an insoluble form in the extracellular matrix. Matrix-localized cellular fibronectin (cFn) contains extra domains (ED1 and/or ED2) not found in plasma Fn (pFn). Very little (< 1-2%) ED1-containing cFn exists in normal blood, and its rapid release into plasma and/or lymph is believed to reflect acute vascular injury.