Glucose 6-phosphate dehydrogenase is regulated through c-Src-mediated tyrosine phosphorylation in endothelial cells.

OBJECTIVE: Glucose 6-phosphate dehydrogenase (G6PD) maintains cellular NADPH levels, which are essential for cellular functions, such as vascular endothelial growth factor (VEGF)-induced angiogenesis. The molecular mechanisms regulating G6PD in angiogenesis are not fully understood. Because tyrosine phosphorylation is a key regulatory pathway for VEGF-mediated endothelial cell (EC) responses, we investigated tyrosine phosphorylation of G6PD and the role of the nonreceptor tyrosine kinase Src. METHODS AND RESULTS: VEGF increased G6PD membrane translocation as measured by a plasma membrane sheet assay, whereas tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyramidine) decreased G6PD translocation by 100%. Furthermore, G6PD tyrosine phosphorylation and plasma membrane activity were increased by VEGF. In resting ECs, tyrosine kinase inhibitors PP2 and herbimycin A decreased basal G6PD activity by approximately 25%, whereas transfection with kinase inactive Src (KD-Src) decreased basal activity by approximately 30%. In mouse embryonic fibroblasts, Src-deficient (SYF) cells showed approximately 22% lower basal G6PD activity than Src-expressing S(+)YF cells. In addition, Src directly phosphorylated G6PD assayed by in vitro kinase assay. In ECs transfected with the G6PD mutants Y428F, Y507F (presumptive sites for Src-phosphorylation) or double mutant Y428F/Y507F, G6PD tyrosine phosphorylation was significantly decreased. Finally, G6PD tyrosine mutants (Y428F, Y507F, and Y428F/Y507F) decreased VEGF-mediated Akt phosphorylation and EC migration. CONCLUSIONS: G6PD activity and membrane association are regulated by Src-mediated tyrosine phosphorylation, which contributes to VEGF-mediated cellular responses in EC.

Thioredoxin in the cardiovascular system.

The thioredoxin (TRX) system (TRX, TRX reductase, and NADPH) is a ubiquitous thiol oxidoreductase system that regulates cellular reduction/oxidation (redox) status. The impairment of cell redox state alters multiple cell pathways, which may contribute to the pathogenesis of cardiovascular disorders including hypertension, atherosclerosis, and heart failure. In this manuscript, we review the essential roles that TRX plays by limiting oxidative stress directly via antioxidant effects and indirectly by protein-protein interactions with key signaling molecules such as thioredoxin interacting protein (TXNIP). TRX and its endogenous regulators may represent important future targets to develop clinical therapies for diseases associated with oxidative stress.

Rho and vascular disease.

The Rho family GTPases are regulatory molecules that link surface receptors to organisation of the actin cytoskeleton and play major roles in fundamental cellular processes. In the vasculature Rho signalling pathways are intimately involved in the regulation of endothelial barrier function, inflammation and transendothelial leukocyte migration, platelet activation, thrombosis and oxidative stress, as well as smooth muscle contraction, migration, proliferation and differentiation, and are thus implicated in many of the changes associated with atherogenesis. Indeed, it is believed that many of the beneficial, non-lipid lowering effects of statins occur as a result of their ability to inhibit Rho protein activation. Conversely, the Rho proteins can have beneficial effects on the vasculature, including the promotion of endothelial repair and the maintenance of SMC differentiation. Further identification of the mechanisms by which these proteins and their effectors act in the vasculature should lead to therapies that specifically target only the adverse effects of Rho signalling.

Leukaemia inhibitory factor retards the progression of atherosclerosis.

OBJECTIVE: Our previous studies showed that the pleiotropic cytokine leukaemia inhibitory factor (LIF) inhibits the de novo formation of experimental atherosclerotic lesions. The present study examined whether LIF also inhibits progression of pre-existing atheroma. METHODS: Balloon angioplasty was performed on the right carotid arteries of 18 rabbits immediately before placing animals on a cholesterol-enriched diet. After 4 weeks, at which time the intima:media ratio (I:M) was 0.99+/-0.12 (n=6), osmotic minipumps containing LIF (n=6) or saline control (n=6) were inserted into the peritoneal cavity of each of the remaining rabbits for a further 4 weeks. Arteries were then harvested for analysis. RESULTS: Continuous administration of LIF for the final 4 weeks of an 8-week cholesterol-enriched diet completely inhibited lesion progression in injured carotid arteries (I:M 1.05+/-0.16) compared with the saline-treated group at 8 weeks (1.62+/-0.13; P<0.05). Similarly in contralateral uninjured carotid arteries, LIF treatment prevented an increase in I:M from a baseline of 0.11+/-0.01 at 4 weeks to 0.15+/-0.02 at 8 weeks compared with 0.40+/-0.04 for the saline-treated group at 8 weeks (P<0.05). LIF reduced the number of macrophages in the neointima of uninjured arteries, but had no effect on the cellular composition of injured arteries. LIF treatment normalised smooth muscle-dependent vasoreactivity to phenylephrine and sodium nitroprusside in both injured and uninjured arteries. Expression and activity of inducible nitric oxide synthase (iNOS) were up-regulated in response to hypercholesterolemia with levels further increased following endothelial denudation. With LIF treatment, iNOS expression was increased in uninjured arteries but marginally reduced in injured arteries. LIF receptors were expressed in both uninjured and injured arteries, with LIF treatment having no significant effect on expression levels. CONCLUSION: LIF prevents progression of pre-formed atherosclerotic plaques, affecting lesion size and vascular reactivity. LIF treatment has differential effects within the artery wall, depending on the presence or absence of de-endothelialisation injury.

Vascular shear stress and activation of inflammatory genes.

Atherosclerotic lesions form preferentially at distinct sites in the arterial tree, especially at or near branch points, bifurcations, and curvatures where there is disturbed or oscillatory blood flow. In contrast, straight regions of the vasculature exhibit uniform laminar shear stress, which is atheroprotective. The ability of laminar flow to exert an anti-inflammatory effect on the endothelial cell lining of the blood vessel is revealed by preventing monocyte adhesion, proliferation, and apoptosis. Changes in endothelial cell gene expression in response to laminar shear stress reflect these changes in cell physiology with the demonstration that physiologic flow inhibits the expression of inflammatory genes. Thus, shear stress is critically important in regulating vascular physiology and pathobiology of the vessel wall via the modulation of endothelial cell gene expression.