Jennifer R. Gamble

High-Density Lipoproteins Inhibit Cytokine-Induced Expression of Endothelial Cell Adhesion Molecules

While an elevated plasma concentration of HDLs is protective against the development of atherosclerosis and ensuing coronary heart disease (CHD), the mechanism of this protection is unknown. One early cellular event in atherogenesis is the adhesion of mononuclear leukocytes to the endothelium. This event is mediated principally by vascular cell adhesion molecule-1 (VCAM-1) but also involves other molecules, such as intercellular adhesion molecule-1 (ICAM-1) and E-selectin. We have investigated the effect of isolated plasma HDLs and reconstituted HDLs on the expression of these molecules by endothelial cells. We show that physiological concentrations of HDLs inhibit tumor necrosis factor-alpha (TNF-alpha) or interleukin-1 (IL-1) induction of these leukocyte adhesion molecules in a concentration-dependent manner. Steady state mRNA levels of TNF-alpha-induced VCAM-1 and E-selectin are significantly reduced by physiological concentrations of HDLs. An an HDL concentration of 1 mg/mL apolipoprotein A-I, the protein expressions of VCAM-1, ICAM-1, and E-selectin were inhibited by 89.6 +/- 0.4% (mean +/-SD, n=4), 64.8 +/- 1.0%, and 79.2 +/- 0.4%, respectively. In contrast, HDLs have no effect on the expression of platelet endothelial cell adhesion molecule (PECAM) or on the expression of the p55 and p75 subunits of the TNF-alpha receptor. HDLs were effective when added from 16 hours before to 5 minutes after cytokine stimulation. HDLs had no effect on TNF-alpha-induced expression of ICAM-1 by human foreskin fibroblasts, suggesting that the effect is cell-type restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiopoietin-1 Is an Antipermeability and Anti-Inflammatory Agent In Vitro and Targets Cell Junctions

Inflammation is a basic pathological mechanism that underlies many diseases. An important component of the inflammatory response is the passage of plasma components and leukocytes from the blood vessel into the tissues. The endothelial monolayer lining blood vessels reacts to stimuli such as thrombin or vascular endothelial growth factor by changes in cell-cell junctions, an increase in permeability, and the leakage of plasma components into tissues. Other stimuli, such as tumor necrosis factor-&agr; (TNF-&agr;), are responsible for stimulating the transmigration of leukocytes. Here we show that angiopoietin-1, a cytokine essential in fetal angiogenesis, not only supports the localization of proteins such as platelet endothelial cell adhesion molecule-1 (PECAM-1) into junctions between endothelial cells and decreases the phosphorylation of PECAM-1 and vascular endothelial cadherin, but it also strengthens these junctions, as evidenced by a decrease in basal permeability and inhibition of permeability responses to thrombin and vascular endothelial growth factor. Furthermore, angiopoietin-1 inhibits TNF-&agr;–stimulated leukocyte transmigration. Angiopoietin-1 may thus have a major role in maintaining the integrity of endothelial monolayers.

An oncogenic role of sphingosine kinase

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

Activation of Sphingosine Kinase by Tumor Necrosis Factor-α Inhibits Apoptosis in Human Endothelial Cells

Human umbilical vein endothelial cells (HUVEC), like most normal cells, are resistant to tumor necrosis factor-α (TNF)-induced apoptosis in spite of TNF activating sphingomyelinase and generating ceramide, a known inducer of apoptosis. Here we report that TNF activates another key enzyme, sphingosine kinase (SphK), in the sphingomyelin metabolic pathway resulting in production of sphingosine-1-phosphate (S1P) and that S1P is a potent antagonist of TNF-mediated apoptosis. The TNF-induced SphK activation is independent of sphingomyelinase and ceramidase activities, suggesting that TNF affects this enzyme directly other than through a mass effect on sphingomyelin degradation. In contrast to normal HUVEC, in a spontaneously transformed endothelial cell line (C11) TNF stimulation failed to activate SphK and induced apoptosis as characterized by morphological and biochemical criteria. Addition of exogenous S1P or increasing endogenous S1P by phorbol ester markedly protected C11 cell line from TNF-induced apoptosis. Conversely,N,N-dimethylsphingosine, an inhibitor of SphK, profoundly sensitized normal HUVEC to killing by TNF. Thus, we demonstrate that the activation of SphK by TNF is an important signaling for protection from the apoptotic effect of TNF in endothelial cells.

High Density Lipoproteins (HDL) Interrupt the Sphingosine Kinase Signaling Pathway A POSSIBLE MECHANISM FOR PROTECTION AGAINST ATHEROSCLEROSIS BY HDL

The ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been demonstrated in their protective function against the development of atherosclerosis and associated coronary heart disease. A key event in atherogenesis is endothelial activation induced by a variety of stimuli such as tumor necrosis factor-α (TNF), resulting in the expression of various adhesion proteins. We have recently reported that sphingosine 1-phosphate, generated by sphingosine kinase activation, is a key molecule in mediating TNF-induced adhesion protein expression. We now show that HDL profoundly inhibit TNF-stimulated sphingosine kinase activity in endothelial cells resulting in a decrease in sphingosine 1-phosphate production and adhesion protein expression. HDL also reduced TNF-mediated activation of extracellular signal-regulated kinases and NF-κB signaling cascades. Furthermore, HDL enhanced the cellular levels of ceramide which in turn inhibits endothelial activation. Thus, the regulation of sphingolipid signaling in endothelial cells by HDL provides a novel insight into the mechanism of protection against atherosclerosis.

Roles of Cyclooxygenase (COX)-1 and COX-2 in Prostanoid Production by Human Endothelial Cells: Selective Up-Regulation of Prostacyclin Synthesis by COX-2

The two cyclooxygenase (COX) isoforms, COX-1 and COX-2, both metabolize arachidonic acid to PGH2, the common substrate for thromboxane A2 (TXA2), prostacyclin (PGI2), and PGE2 synthesis. We characterized the synthesis of these prostanoids in HUVECs in relation to COX-1 and COX-2 activity. Untreated HUVEC expressed only COX-1, whereas addition of IL-1β caused induction of COX-2. TXA2 was the predominant COX-1-derived product, and TXA2 synthesis changed little with up-regulation of COX-2 by IL-1β (2-fold increase). By contrast, COX-2 up-regulation was associated with large increases in the synthesis of PGI2 and PGE2 (54- and 84-fold increases, respectively). Addition of the selective COX-2 inhibitor, NS-398, almost completely abolished PGI2 and PGE2 synthesis, but had little effect on TXA2 synthesis. The up-regulation of COX-2 by IL-1β was accompanied by specific up-regulation of PGI synthase and PGE synthase, but not TX synthase. An examination of the substrate concentration dependencies showed that the pathway of TXA2 synthesis was saturated at a 20-fold lower arachidonic acid concentration than that for PGI2 and PGE2 synthesis. In conclusion, endothelial prostanoid synthesis appears to be differentially regulated by the induction of COX-2. The apparent PGI2 and PGE2 linkage with COX-2 activity may be explained by a temporal increase in total COX activity, together with selective up-regulation of PGI synthase and PGE synthase, and different kinetic characteristics of the terminal synthases. These findings have particular importance with regard to the potential for cardiovascular consequences of COX-2 inhibition.

Expression of a Catalytically Inactive Sphingosine Kinase Mutant Blocks Agonist-induced Sphingosine Kinase Activation A DOMINANT-NEGATIVE SPHINGOSINE KINASE

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly82 to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-α (TNFα), interleukin-1β, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSKG82D). Basal SK activity was unaffected by expression of hSKG82D. Expression of hSKG82D had no effect on TNFα-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSKG82D acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNFα activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

ANGIOGENESIS: MODELS AND MODULATORS

Angiogenesis in vivo is distinguished by four stages: subsequent to the transduction of signals to differentiate, stage 1 is defined as an altered proteolytic balance of the cell allowing it to digest through the surrounding matrix. These committed cells then proliferate (stage 2), and migrate (stage 3) to form aligned cords of cells. The final stage is the development of vessel patency (stage 4), generated by a coalescing of intracellular vacuoles. Subsequently, these structures anastamose and the initial flow of blood through the new vessel completes the process. We present and discuss how the available models most closely represent phases of in vivo angiogenesis. The enhancement of angiogenesis by hyaluronic acid fragments, transforming growth factor beta, tumor necrosis factor alpha, angiogenin, okadaic acid, fibroblast growth factor, interleukin 8, vascular endothelial growth factor, haptoglobin, and gangliosides, and the inhibition of the process by hyaluronic acid, estrogen metabolites, genestein, heparin, cyclosporin A, placental RNase inhibitor, steroids, collagen synthesis inhibitors, thrombospondin, fumagellin, and protamine are also discussed.

Factors Influencing the Ability of HDL to Inhibit Expression of Vascular Cell Adhesion Molecule-1 in Endothelial Cells

We have previously reported that high density lipoproteins (HDLs) inhibit the cytokine-induced expression of adhesion molecules in endothelial cells. Here we investigate whether different preparations of HDLs vary in their ability to inhibit the expression of vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVECs) activated by tumor necrosis factor-alpha (TNF-alpha). HDLs collected from a number of different human subjects all inhibited VCAM-1 expression in a concentration-dependent manner, although the extent of inhibition varied widely between subjects. The inhibitory activities of the HDL2 and HDL3 subfractions isolated from individual subjects also differed. Whether equated for concentrations of apolipoprotein (apo) A-I or cholesterol, the inhibitory activity of HDL3 was superior to that of HDL2. This difference remained apparent even when the HDL subfractions were present only during preincubations with the HUVECs and were removed before activation by TNF-alpha. To determine whether the inhibitory effect of HDL3 was influenced by apolipoprotein composition, preparations of HDL3 were modified by replacing all of their apo A-I with apo A-II. This change in apolipoprotein composition had no effect on the ability of the HDL3 to inhibit endothelial VCAM-1 expression. Thus, it has been shown that different preparations of HDLs differ markedly in their abilities to inhibit VCAM-1 expression in cytokine-activated HUVECs. The mechanism underlying the differences remains to be determined.

Cloning, Characterization, and Chromosomal Location of a Novel Human K+-Cl− Cotransporter

Differential display polymerase chain reaction has been used to isolate genes regulated in vascular endothelial cells by the angiogenic factor vascular endothelial cell growth factor (VEGF). Analysis of one of the bands consistently up-regulated by VEGF led us to the identification of a cDNA from a human umbilical vein endothelial cell library that is 77% identical to the human K+-Cl− cotransporter1 (KCC1). We have referred to the predicted protein as K+-Cl−cotransporter 3 (KCC3). Hydrophobicity analysis of the KCC3 amino acid sequence showed an almost identical pattern to KCC1, suggesting 12 membrane-spanning segments, a large extracellular loop with potentialN-glycosylation sites, and cytoplasmic N- and C-terminal regions. The KCC3 mRNA was highly expressed in brain, heart, skeletal muscle, and kidney, showing a distinct pattern and size from KCC1 and KCC2. The KCC3 mRNA level in endothelial cells increased on treatment with VEGF and decreased with the proinflammatory cytokine tumor necrosis factor α, whereas KCC1 mRNA levels remained unchanged. Stable overexpression of KCC3 cDNA in HEK293 cells produced a glycoprotein of approximately 150 kDa, which was reduced to 120 kDa by glycosidase digestion. An increased initial uptake rate of86Rb was seen in clones with high KCC3 expression, which was dependent on extracellular Cl− but not Na+and was inhibitable by the loop diuretic agent furosemide. The KCC3 genomic localization was shown to be 15q13 by fluorescence in situ hybridization. Radiation hybrid analysis placed KCC3 within an area associated with juvenile myoclonic epilepsy. These results suggest KCC3 is a new member of the KCC family that is under distinct regulation from KCC1.