Caroline P.D. Wheeler-Jones

Vascular endothelial growth factor stimulates prostacyclin production and activation of cytosolic phospholipase A2 in endothelial cells via p42/p44 mitogen-activated protein kinase

Vascular endothelial growth factor (VEGF) stimulated a time‐ and concentration‐dependent increase in PGI2 synthesis in human umbilical vein endothelial cells with a mean maximum increase of 2‐fold above basal levels at 25 ng/ml after 60 min. VEGF also rapidly stimulated the release of arachidonic acid and phosphorylation and activation of cytosolic phospholipase A2 (cPLA2). The VEGF‐related factor, placenta growth factor (PlGF), had little effect on PGI2 synthesis, arachidonic acid release or cPLA2 activation. PD98059, a selective inhibitor of MAP kinase kinase, caused complete inhibition of VEGF‐stimulated MAP kinase activity, PGI2 synthesis and cPLA2 gel retardation, but had no effect on VEGF‐induced vWF secretion. These findings provide the first evidence that VEGF can stimulate PGI2 synthesis via cPLA2‐mediated arachidonic acid release and indicate that VEGF stimulation of this biosynthetic pathway may occur, at least in part, via activation of p42/p44 MAP kinases.

Effects of protein tyrosine kinase inhibitors on cytokine-induced adhesion molecule expression by human umbilical vein endothelial cells

1 . Endothelial cells can be stimulated by the pro‐inflammatory cytokines interleukin (IL)‐1α and tumour necrosis factor (TNF)α to express the leukocyte adhesion molecules E‐selectin, vascular cell adhesion molecule (VCAM)‐1 and intercellular adhesion molecule (ICAM)‐1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine‐activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2 . Maximal E‐selectin expression induced by incubation of HUVEC for 4 h with IL‐1α (100 u ml−1) and TNFα (100 u ml−1) was dose‐dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12‐myristate, 13‐acetate (PMA)‐induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31‐7549 or Ro31‐8220 did not affect IL‐1α‐ or TNFα‐induced E‐selectin expression at concentrations which maximally inhibited PMA‐induced expression. 3 . Genistein inhibited VCAM‐1 expression induced by incubation of HUVEC for 24 h with TNFα or IL‐1α whereas it did not affect ICAM‐1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM‐1 and ICAM‐1 expression induced by TNFα. 4 . Basal expression of E‐selectin, VCAM‐1 and ICAM‐1 was dose‐dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNFα‐induced expression of these molecules with maximal E‐selectin and ICAM‐1 expression being slightly enhanced and VCAM‐1 expression dose‐dependently reduced. 5 . We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre‐myeloid cell line U937 to TNFα‐stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNFα was dose‐dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E‐selectin and VCAM‐1 but after 24 h was only inhibited by anti‐VCAM‐1. 6 . Sodium orthovanadate had no effect on TNFα‐induced U937 adhesion but dose‐dependently enhanced adhesion to unstimulated HUVEC. Vanadate‐induced adhesion was inhibited by an antibody against VCAM‐1. 7 . These results demonstrate that PTK‐mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine‐induced adhesion molecule expression.

Mastoparan stimulates insulin secretion from pancreatic β-cells by effects at a late stage in the secretory pathway

Mastoparan (MP) is a component of wasp venom which stimulates secretion from a number of cell types. We have used intact and electrically permeabilised islets of Langerhans to investigate the mechanisms through which MP stimulates insulin secretion from pancreatic beta-cells. MP caused a temperature-dependent and dose-related stimulation of insulin secretion from intact islets at a substimulatory concentration (2 mM) of glucose, which was not dependent upon the presence of extracellular Ca2+. MP also stimulated ATP-independent insulin secretion from electrically permeabilised islets in which intracellular Ca2+ was clamped at a substimulatory concentration (50 nM). MP-induced insulin secretion was not inhibited by down-regulation of islet protein kinase C, nor by the protein kinase inhibitor staurosporine, nor by the cyclic AMP antagonist Rp-adenosine 3,5-cyclic phosphorothioate. However, MP-induced secretion from permeabilised islets was inhibited by the presence of guanosine 5-O-2-thiodiphosphate. These results suggest that MP stimulates insulin secretion by a mechanism that is independent of changes in cytosolic Ca2+ or protein kinase activation, but which is dependent, at least in part, upon activation of a GTP-binding protein at a late stage in the secretory process.

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti‐inflammatory derivatives with gastrointestinalsparing properties

1 The effects of novel nitric oxide‐releasing nonsteroidal anti‐inflammatory compounds (NO‐NSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line, J774. 2 LPS‐induced nitrite production was markedly attenuated by the nitroxybutylester derivatives of flurbiprofen (FNBE), aspirin, ketoprofen, naproxen, diclofenac and ketorolac, with each compound reducing accumulated nitrite levels by >40% at the maximum concentrations (100 μg ml−1) used. 3 Further examination revealed that nitrite production was inhibited in a concentration‐dependent (1–100 μg ml−1) manner by FNBE which at 100 μg ml−1 decreased LPS‐stimulated levels by 63.3±8.6% (n=7). The parent compound flurbiprofen was relatively ineffective over the same concentration‐range, inhibiting nitrite accumulation by 24±0.9% (n=3) at the maximum concentration used (100 μg ml−1). 4 FNBE reduced LPS‐induced nitrite production when added to cells up to 4 h after LPS. Thereafter, FNBE caused very little or no reduction in nitrite levels. Furthermore NO‐NSAIDs (100 μg ml−1) did not inhibit the metabolism of L‐[3H]‐arginine to citrulline by NO synthase isolated from LPS‐activated macrophages. 5 Western blot analysis demonstrated that NO synthase expression was markedly attenuated following co‐incubation of J774 cell with LPS (1 μg ml−1; 24 h) and FNBE (100μg ml−1; 24 h). Thus taken together, these findings indicate that NO‐NSAIDs inhibit induction of NO synthase without directly affecting enzyme activity. 6 In conclusion our results indicate that NO‐NSAIDs can inhibit the inducible L‐arginine‐NO pathway, and are capable of suppressing NO synthesis by inhibiting expression of NO synthase. The clinical implications of these findings remain to be established.

Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E-selectin expression

We have examined the potential role of MAP kinase in the regulation of endothelial cell PGI2 synthesis, vWF secretion and E‐selectin expression using the specific MEK inhibitor PD98059. PD98059 dose‐dependently attenuated the tyrosine phosphorylation and activation of p42mapk in response to thrombin or inflammatory cytokines. Inhibition of thrombin‐induced p42mapk activation was paralleled by an inhibitory effect of PD98059 on thrombin‐driven PGI2 generation but not on vWF secretion or IL‐1α/TNFα‐induced E‐selectin expression. These results provide evidence for a key role for p42mapk in the acute regulation of PGI2 synthesis in human endothelial cells and suggest that activation of the MAP kinase cascade is not obligatory for cytokine‐stimulated E‐selectin expression.

Activation of p42mapk in human umbilical vein endothelial cells by interleukin-1α and tumor necrosis factor-α

Work from this and other laboratories has identified a role for protein tyrosine kinases in interleukin-1α (IL-1α)- and tumor necrosis factor-α (TNF-α)-induced responses in endothelial cells. In this study, we show that activation of human umbilical vein endothelial cells (HUVEC) by IL-1α leads to increased tyrosine phosphorylation of several proteins including one with a molecular mass of ∼42 kDa. This protein was identified as p42mapk by Western blot analysis. Tyrosine phosphorylation and catalytic activation of p42mapk by IL-1α was transient, reaching maximal levels after 30 min and returning to basal levels by 120-300 min. Activation of p42mapk in HUVEC was also observed in response to TNF-α or to the protein kinase C (PKC)-activating phorbol ester phorbol 12-myristate 13-acetate (PMA). Pretreatment of HUVEC with IL-1α or TNF-α prevented reactivation of p42mapk by either cytokine but did not affect subsequent activation in response to PMA. Activation of p42mapk by PMA was significantly reduced by the PKC inhibitor Ro-31-8220 and completely inhibited by the protein tyrosine kinase inhibitor genistein. Genistein, but not Ro-31-8220, attenuated IL-1α- and TNF-α-induced p42mapk activation. Taken together, the results of this study demonstrate 1) that p42mapk is transiently activated in HUVEC by IL-1α and TNF-α, 2) that this activation is PKC independent, and 3) that a genistein-inhibitable tyrosine kinase may be an upstream regulator of cytokine-induced p42mapk activation in human endothelium.

High-fat meals rich in EPA plus DHA compared with DHA only have differential effects on postprandial lipemia and plasma 8-isoprostane F2α concentrations relative to a control high–oleic acid meal: a randomized controlled trial

Background: Eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) supplementation has beneficial cardiovascular effects, but postprandial influences of these individual fatty acids are unclear. Objectives: The primary objective was to determine the vascular effects of EPA + DHA compared with DHA only during postprandial lipemia relative to control high–oleic acid meals; the secondary objective was to characterize the effects of linoleic acid–enriched high-fat meals relative to the control meal. Design: We conducted a randomized, controlled, double-blind crossover trial of 4 high-fat (75-g) meals containing 1) high–oleic acid sunflower oil (HOS; control), 2) HOS + fish oil (FO; 5 g EPA and DHA), 3) HOS + algal oil (AO; 5 g DHA), and 4) high–linoleic acid sunflower oil (HLS) in 16 healthy men (aged 35–70 y) with higher than optimal fasting triacylglycerol concentrations (mean ± SD triacylglycerol, 1.9 ± 0.5 mmol/L). Results: Elevations in triacylglycerol concentration relative to baseline were slightly reduced after FO and HLS compared with the HOS control (P < 0.05). The characteristic decrease from baseline in plasma nonesterified fatty acids after a mixed meal was inhibited after AO (Δ 0–3 h, P < 0.05). HLS increased the augmentation index compared with the other test meals (P < 0.05), although the digital volume pulse–reflection index was not significantly different. Plasma 8-isoprostane F2α analysis revealed opposing effects of FO (increased) and AO (reduced) compared with the control (P < 0.05). No differences in nitric oxide metabolites were observed. Conclusions: These data show differential postprandial 8-isoprostane F2α responses to high-fat meals containing EPA + DHA–rich fish oil compared with DHA-rich AO, but these differences were not associated with consistent effects on postprandial vascular function or lipemia. More detailed analyses of polyunsaturated fatty acid–derived lipid mediators are required to determine possible divergent functional effects of single meals rich in either DHA or EPA. This trial was registered at clinicaltrials.gov as NCT01618071.

High-fat meals rich in EPA plus DHA compared with DHA only have differential effects on postprandial lipemia and plasma 8-isoprostane F2α concentrations compared to a control high-oleic acid meal

Background: Eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) supplementation has beneficial cardiovascular effects, but postprandial influences of these individual fatty acids are unclear. Objectives: The primary objective was to determine the vascular effects of EPA + DHA compared with DHA only during postprandial lipemia relative to control high–oleic acid meals; the secondary objective was to characterize the effects of linoleic acid–enriched high-fat meals relative to the control meal. Design: We conducted a randomized, controlled, double-blind crossover trial of 4 high-fat (75-g) meals containing 1) high–oleic acid sunflower oil (HOS; control), 2) HOS + fish oil (FO; 5 g EPA and DHA), 3) HOS + algal oil (AO; 5 g DHA), and 4) high–linoleic acid sunflower oil (HLS) in 16 healthy men (aged 35–70 y) with higher than optimal fasting triacylglycerol concentrations (mean ± SD triacylglycerol, 1.9 ± 0.5 mmol/L). Results: Elevations in triacylglycerol concentration relative to baseline were slightly reduced after FO and HLS compared with the HOS control (P < 0.05). The characteristic decrease from baseline in plasma nonesterified fatty acids after a mixed meal was inhibited after AO (Δ 0–3 h, P < 0.05). HLS increased the augmentation index compared with the other test meals (P < 0.05), although the digital volume pulse–reflection index was not significantly different. Plasma 8-isoprostane F2α analysis revealed opposing effects of FO (increased) and AO (reduced) compared with the control (P < 0.05). No differences in nitric oxide metabolites were observed. Conclusions: These data show differential postprandial 8-isoprostane F2α responses to high-fat meals containing EPA + DHA–rich fish oil compared with DHA-rich AO, but these differences were not associated with consistent effects on postprandial vascular function or lipemia. More detailed analyses of polyunsaturated fatty acid–derived lipid mediators are required to determine possible divergent functional effects of single meals rich in either DHA or EPA. This trial was registered at clinicaltrials.gov as NCT01618071.

High-fat meals rich in EPA plus DHA compared with DHA only have differential effects on postprandial lipemia and plasma 8-isoprostane F2α concentrations compared to a control high-oleic acid meal: a randomized controlled trial

Background: Eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) supplementation has beneficial cardiovascular effects, but postprandial influences of these individual fatty acids are unclear. Objectives: The primary objective was to determine the vascular effects of EPA + DHA compared with DHA only during postprandial lipemia relative to control high–oleic acid meals; the secondary objective was to characterize the effects of linoleic acid–enriched high-fat meals relative to the control meal. Design: We conducted a randomized, controlled, double-blind crossover trial of 4 high-fat (75-g) meals containing 1) high–oleic acid sunflower oil (HOS; control), 2) HOS + fish oil (FO; 5 g EPA and DHA), 3) HOS + algal oil (AO; 5 g DHA), and 4) high–linoleic acid sunflower oil (HLS) in 16 healthy men (aged 35–70 y) with higher than optimal fasting triacylglycerol concentrations (mean ± SD triacylglycerol, 1.9 ± 0.5 mmol/L). Results: Elevations in triacylglycerol concentration relative to baseline were slightly reduced after FO and HLS compared with the HOS control (P < 0.05). The characteristic decrease from baseline in plasma nonesterified fatty acids after a mixed meal was inhibited after AO (Δ 0–3 h, P < 0.05). HLS increased the augmentation index compared with the other test meals (P < 0.05), although the digital volume pulse–reflection index was not significantly different. Plasma 8-isoprostane F2α analysis revealed opposing effects of FO (increased) and AO (reduced) compared with the control (P < 0.05). No differences in nitric oxide metabolites were observed. Conclusions: These data show differential postprandial 8-isoprostane F2α responses to high-fat meals containing EPA + DHA–rich fish oil compared with DHA-rich AO, but these differences were not associated with consistent effects on postprandial vascular function or lipemia. More detailed analyses of polyunsaturated fatty acid–derived lipid mediators are required to determine possible divergent functional effects of single meals rich in either DHA or EPA. This trial was registered at clinicaltrials.gov as NCT01618071.