An endogenous inhibitor of angiogenesis downregulated by hypoxia in human aortic valve stenosis promotes disease pathogenesis.

Aortic valve stenosis is the most common valve disease in the western world. Central to the pathogenesis of this disease is the growth of new blood vessels (angiogenesis) within the aortic valve allowing infiltration of immune cells and development of intra-valve inflammation. Identifying the cellular mediators involved in this angiogenesis is important as this may reveal new therapeutic targets which could ultimately prevent the progression of aortic valve stenosis. Aortic valves from patients undergoing surgery for aortic valve replacement or dilation of the aortic arch were examined both ex vivo and in vitro. We now demonstrate that the anti-angiogenic protein, soluble fms-like tyrosine kinase 1 (sFlt1), a non-signalling soluble receptor for vascular endothelial growth factor, is constitutively expressed in non-diseased valves. sFlt-1 expression was, however, significantly reduced in aortic valve tissue from patients with aortic valve stenosis while protein markers of hypoxia were simultaneously increased. Exposure of primary-cultured valve interstitial cells to hypoxia resulted in a decrease in the expression of sFlt-1. We further reveal using a bioassay that siRNA knock-down of sFlt1 in valve interstitial cells directly results in a pro-angiogenic environment. Finally, incubation of aortic valves with sphingosine 1-phosphate, a bioactive lipid-mediator, increased sFlt-1 expression and inhibited angiogenesis within valve tissue. In conclusion, this study demonstrates that sFlt1 expression is directly correlated with angiogenesis in aortic valves and the observed decrease in sFlt-1 expression in aortic valve stenosis could increase valve inflammation, promoting disease progression. This could be a viable therapeutic target in treating this disease.

Sphingosine-1-phosphate-induced release of TIMP-2 from vascular smooth muscle cells inhibits angiogenesis.

Following myocardial infarction, angiogenesis occurs as a result of thrombus formation, which permits reperfusion of damaged myocardium. Sphingosine 1-phosphate (S1P) is a naturally occurring lipid mediator released from platelets and is found in high concentrations at sites of thrombosis. S1P might therefore be involved in regulating angiogenesis following myocardial infarction and might influence reperfusion. The aims of this study were to determine the effects of S1P in human coronary arterial cell angiogenesis and delineate the subsequent mechanisms. An in vitro model of angiogenesis was developed using a co-culture of human coronary artery endothelial cells, human coronary smooth muscle cells and human fibroblasts. In this model, S1P inhibited angiogenesis and this was dependent on the presence of smooth muscle cells. The mechanism of the inhibitory effect was through S1P-induced release of a soluble mediator from smooth muscle cells. This mediator was identified as tissue inhibitor of metalloproteinase-2 (TIMP-2). Release of TIMP-2 was dependent on S1P-induced activation of Rho kinase and directly contributed to incomplete formation of endothelial cell adherens junctions. This was observed as a diffuse localisation of VE-cadherin, leading to decreased tubulogenesis. A similar inhibitory response to S1P was demonstrated in an ex vivo human arterial model of angiogenesis. In summary, S1P-induced inhibition of angiogenesis in human artery endothelial cells is mediated by TIMP-2 from vascular smooth muscle cells. This reduces the integrity of intercellular junctions between nascent endothelial cells. S1P might therefore inhibit the angiogenic response following myocardial infarction.

Plasma zinc's alter ego is a low-molecular-weight humoral factor.

Mild dietary zinc deprivation in humans and rodents has little effect on blood plasma zinc levels, and yet cellular consequences of zinc depletion can be detected in vascular and other tissues. We proposed that a zinc-regulated humoral factor might mediate the effects of zinc deprivation. Using a novel approach, primary rat vascular smooth muscle cells (VSMCs) were treated with plasma from zinc-deficient (<1 mg Zn/kg) or zinc-adequate (35 mg Zn/kg, pair-fed) adult male rats, and zinc levels were manipulated to distinguish direct and indirect effects of plasma zinc. Gene expression changes were analyzed by microarray and qPCR, and incubation of VSMCs with blood plasma from zinc-deficient rats strongly changed the expression of >2500 genes, compared to incubation of cells with zinc-adequate rat plasma. We demonstrated that this effect was caused by a low-molecular-weight (∼2-kDa) zinc-regulated humoral factor but that changes in gene expression were mostly reversed by adding zinc back to zinc-deficient plasma. Strongly regulated genes were overrepresented in pathways associated with immune function and development. We conclude that zinc deficiency induces the production of a low-molecular-weight humoral factor whose influence on VSMC gene expression is blocked by plasma zinc. This factor is therefore under dual control by zinc.

Microanalytical isotope ratio measurements and elemental mapping using laser ablation ICP-MS for tissue thin sections: zinc tracer studies in rats.

The kinetics of zinc absorption, metabolism and excretion is extensively studied by nutritionists. Stable isotopes of zinc can be used to identify body zinc compartments that have different turnover kinetics. Since the compartments might belong to physiological subsections of different organs, there is a need for microsampling analysis to determine isotope ratios of the trace element zinc in tissue samples. Here, we study the feasibility to use laser ablation coupled to quadrupole ICP-MS for the determination of zinc tracers given to rats at different time points with the aim to generate isotope ratio bioimages of heart tissue. A double tracer ((70)Zn and (67)Zn) experiment with rats was designed to label the exchangeable zinc pool as well as the stable zinc pool. The isotope ratios determined by laser ablation ICP-MS were evaluated by additional measurements of tissue digests. Accumulated tracers which made up more than 0.1% of total zinc could be identified in the tissues of the treated rats. It was established that at least 50 measurements from the microsampling were necessary to distinguish between controls and a tracer treated rat resulting in reduced resolution of the bioimage. With the parameters used, features in the tissue thin sections of at least 250 µm(2) in size are necessary to detect the incorporation of a tracer. When different time points have to be measured, higher precisions are required and therefore a larger area needs to be ablated (1 mm(2)). Using the bioimages and pool measurements from one physiological feature, it was possible to show that the aorta cell walls incorporate the zinc tracer at the different time points.

Phosphoprotein enriched in astrocytes (PEA)-15 is a novel regulator of adipose tissue expansion.

Excessive expansion of adipose tissue in obesity typically leads to overflow and accumulation of lipids in other tissues, causing fatty liver disease and atherosclerosis. The intracellular protein, phosphoprotein enriched in astrocytes (PEA)-15 has been linked to metabolic disease but its role in lipid storage has not been examined. To delineate the role of PEA-15 in adipose tissue, we placed PEA-15-/- mice on a high fat diet. These mice developed increased body weight and greater white adipose tissue expansion compared to high fat diet-fed wild type mice. This was due to increased adipocyte cell size in PEA-15-/- mice consistent with greater lipid storage capacity. Surprisingly, PEA-15-/- mice exhibited improvements in whole body insulin sensitivity, lower hepatic weight and decreased serum triglycerides indicating a protective phenotype. To determine effects on atherosclerosis, PEA-15-/- mice were crossed with the ApoE-/- mice on a high fat diet. Strikingly, these mice were protected from atherosclerosis and had less hepatic lipid accumulation despite increased adiposity. Therefore, we reveal for the first time that PEA-15 plays a novel role in regulating the expansion of adipose tissue. Decreasing PEA-15 expression increases the sequestering of lipids in adipose tissue, protecting other tissues in obesity, thereby improving metabolic health.

The multi-functional role of sphingosylphosphorylcholine.

The sphingomyelin metabolite, sphingosylphosphorylcholine (SPC) has been the subject of much recent interest and controversy. Studies have indicated that SPC naturally occurs in plasma and a constituent of lipoproteins. Synthesis is also increased in some pathological conditions. Research has demonstrated that SPC is a potentially important lipid mediator of cell type specific functions in major tissues, such as heart, blood vessels, skin, brain and immune system. These effects are regulated via a number of different intracellular signalling cascades, also dependent upon cell type. Initial reports identifying high affinity SPC receptors at first appeared to reinforce the physiological relevance of this sphingolipid. However, these studies have now been retracted. Some SPC effects have been shown be occur via plasma membrane receptors for the related sphingolipid, sphingosine 1-phosphate (S1P). Despite a lack of well-defined receptor signal transduction mechanisms and sparse pharmacological data, several key characteristics of SPC are now emerging. SPC can act as a mitogen in several different cell types and in certain circumstances, may also be a pro-inflammatory mediator. In this review, these actions of SPC are discussed with a view to understanding the potential physiological relevance of this sphingolipid.

Randomized controlled trial of aspirin and clopidogrel versus aspirin and placebo on markers of smooth muscle proliferation before and after peripheral angioplasty.

OBJECTIVE: In peripheral arterial disease (PAD) patients, a limiting factor in the success of percutaneous transluminal angioplasty (PTA) is the development of restenosis secondary to vascular smooth muscle cell (SMC) proliferation. Following endothelial damage and platelet activation, there is release of factors and adhesion molecules which affect SMC proliferation. The aim of this study was to determine the effect of combination antiplatelet therapy (clopidogrel and aspirin compared with aspirin and placebo) on the ability of plasma from PAD patients undergoing PTA to stimulate SMCs in vitro. We further aimed to investigate the effect of combination treatment on the levels of circulating adhesion molecules and factors, which are known to mediate SMC proliferation in experimental models. METHODS: Fifty patients were randomized to receive blinded clopidogrel or placebo, for thirty days, in addition to their daily 75 mg aspirin. To measure proliferative capacity, diluted plasma was incubated for 15 minutes with 24 hour-growth-arrested rat vascular smooth muscle cells, and extracellular regulated kinase (ERK)1/2 activation was analyzed by Western blotting at baseline, one hour pre-PTA, one hour, 24 hours and 30 days post-PTA. Plasma platelet-derived growth factor (PDGF), sE-selectin, intracellular adhesion molecule-1 (sICAM-1), and von Willebrand factor (vWF) were measured by ELISA, at the same five timepoints. Platelet activation was measured by flow cytometry of ADP-stimulated platelet fibrinogen binding at baseline and one hour post-PTA. RESULTS: ADP-stimulated platelet fibrinogen binding was significantly inhibited by clopidogrel before and after PTA. ERK 1/2 activation was significantly increased post-PTA in both the aspirin/clopidogrel and aspirin/placebo groups (P < .001). There was a statistically significant decrease in PDGF (P = .004), and increase in vWF (P = .026), following loading with clopidogrel. sICAM-1 levels significantly decreased (P = .016) in the aspirin/placebo group following PTA. There were no other significant changes and also there was no statistically significant difference between the two treatment groups for each of ERK 1/2, sICAM-1, sE-selectin, or vWF. CONCLUSIONS: This is the first study to show in-vitro ERK 1/2 activation (a surrogate marker of SMC proliferation) increases post-PTA. Combination antiplatelet therapy had no significant effect on this, although it did reduce PDGF. Further work is required to evaluate potential therapeutic treatments, which may reduce peripheral PTA-induced smooth muscle cell activation. CLINICAL RELEVANCE: High rates of restenosis remain the major limitation of peripheral arterial angioplasty and stenting.The restenotic lesion occurs secondary to platelet activation, released circulating factors, and subsequent smooth musclecell proliferation and migration into the intima. Methods to limit the restenotic lesion are poorly understood. This paperinvestigates the effect of PTA on smooth muscle cell activation and the release of factors in plasma which mediate SMCproliferation. It also examines the effect of combination antiplatelet therapy as a potential therapeutic strategy.

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle.

In arterial myocytes the Ca(2+) mobilizing messenger NAADP evokes spatially restricted Ca(2+) bursts from a lysosome-related store that are subsequently amplified into global Ca(2+) waves by Ca(2+)-induced Ca(2+)-release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) abolished global Ca(2+) waves but not Ca(2+) bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca(2+) signalling by NAADP.

PEA-15 (Phosphoprotein Enriched in Astrocytes 15) Is a Protective Mediator in the Vasculature and Is Regulated During Neointimal Hyperplasia.

BACKGROUND: Neointimal hyperplasia following angioplasty occurs via vascular smooth muscle cell proliferation. The mechanisms involved are not fully understood but include mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2). We recently identified the intracellular mediator PEA-15 (phosphoprotein enriched in astrocytes 15) in vascular smooth muscle cells as a regulator of ERK1/2-dependent proliferation in vitro. PEA-15 acts as a cytoplasmic anchor for ERK1/2, preventing nuclear localization and thereby reducing ERK1/2-dependent gene expression. The aim of the current study was to examine the role of PEA-15 in neointimal hyperplasia in vivo. METHOD AND RESULTS: Mice deficient in PEA-15 or wild-type mice were subjected to wire injury of the carotid artery. In uninjured arteries from PEA-15-deficient mice, ERK1/2 had increased nuclear translocation and increased basal ERK1/2-dependent transcription. Following wire injury, arteries from PEA-15-deficient mice developed neointimal hyperplasia at an increased rate compared with wild-type mice. This occurred in parallel with an increase in a proliferative marker and vascular smooth muscle cell proliferation. In wild-type mice, PEA-15 expression was decreased in vascular smooth muscle cells at an early stage before any increase in intima:media ratio. This regulation of PEA-15 expression following injury was also observed in an ex vivo human model of hyperplasia. CONCLUSIONS: These results indicate, for the first time, a novel protective role for PEA-15 against inappropriate vascular proliferation. PEA-15 expression may also be repressed during vascular injury, suggesting that maintenance of PEA-15 expression is a novel therapeutic target in vascular disease.

Sphingolipids differentially regulate mitogen-activated protein kinases and intracellular Ca2+ in vascular smooth muscle: effects on CREB activation.

1. Related sphingolipids, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC), have important effects on vascular smooth muscle. The aim of this study was to investigate the intracellular pathways regulated by S1P and SPC in rat cerebral artery. 2. In cerebral arteries, S1P increased extracellular signal-regulated kinase (ERK)1/2 phosphorylation (5.2+/-1.4-fold increase) but did not activate p38 mitogen-activated protein kinase (p38MAPK) as assessed by immunoblotting. In contrast, SPC increased p38MAPK phosphorylation (3.0+/-0.3-fold increase) but did not stimulate ERK1/2. This differential activation was confirmed by measuring activation of heat shock protein (HSP) 27, a known downstream target of p38MAPK. Only SPC, but not S1P, activated HSP27. 3. In enzymatically dispersed cerebral artery myocytes, SPC increased [Ca2+]i in a concentration-dependent manner (peak response at 10 microM: 0.4+/-0.02 ratio units) as determined using the Ca2+ indicator, Fura 2. In contrast to S1P, the SPC-induced [Ca2+]i increase did not involve intracellular release but was due to Ca2+ influx via L-type Ca2+ channels. 4. Despite differences in signalling, both S1P and SPC phosphorylated the transcription factor cAMP response element-binding protein (CREB). S1P-induced CREB activation was dependent on ERK1/2 and Ca2+-calmodulin-dependent protein kinase (CaMK) activation. CREB activation by SPC required both p38MAPK and CaMK activation, but not ERK1/2. 5. In conclusion, S1P and SPC activate distinct MAP kinase isoforms and increase [Ca2+]i via different mechanisms in rat cerebral artery. This does not affect the ability of S1P or SPC to activate CREB, although this occurs via different pathways.

Comparison of sphingosine 1-phosphate-induced intracellular signaling pathways in vascular smooth muscles: differential role in vasoconstriction.

Sphingosine 1-phosphate (S1P), a lipid released from activated platelets, influences physiological processes in the cardiovascular system via activation of the endothelial differentiation gene (EDG/S1P) family of 7 transmembrane G protein-coupled receptors. In cultured vascular smooth muscle (VSM) cells, S1P signaling has been shown to stimulate proliferative responses; however, its role in vasoconstriction has not been examined. In the present study, the effects of S1P and EDG/S1P receptor expression were determined in rat VSM from cerebral artery and aorta. S1P induced constriction of cerebral artery, which was partly dependent on activation of p160(ROCK) (Rho-kinase). S1P also induced activation of RhoA in cerebral artery with a similar time course to contraction. In aorta, S1P did not produce a constriction or RhoA activation. In VSM myocytes from cerebral arteries, stimulation with S1P gives rise to a global increase in [Ca2+]i, initially generated via Ca2+ release from the sarcoplasmic reticulum by an inositol 1,4,5-trisphosphate-dependent pathway. In aorta VSM, a small increase in [Ca2+]i was observed after stimulation at higher concentrations of S1P. S1P induced activation of p42/p44(mapk) in aorta and cerebral artery VSM. Subtype-specific S1P receptor antibodies revealed that the expression of S1P3/EDG-3 and S1P2/EDG-5 receptors is 4-fold higher in cerebral artery compared with aorta. S1P(1)/EDG-1 receptor expression was similar in both types of VSM. Therefore, the ability of S1P to act as a vasoactive mediator is dependent on the activation of associated signaling pathways and may vary in different VSM. This differential signaling may be related to the expression of S1P receptor subtypes.

PDGF-induced signaling in proliferating and differentiated vascular smooth muscle: effects of altered intracellular Ca2+ regulation.

OBJECTIVE: Platelet-derived growth factor-BB (PDGF)-induced intracellular signaling is involved in phenotypic modulation of vascular smooth muscle (VSM). This study has examined the PDGF-induced Ca2+ increase and the resultant effect on signaling pathways in proliferative compared with fully differentiated VSM. METHODS: PDGF-induced changes in Ca2+ were measured in portal vein (PV) myocytes from 2-4-day-old (proliferating), compared to 6-week-old (differentiated), Sprague Dawley rats. Phospholipase C (PLC)gamma expression and activation of extracellular signal-regulated kinase (ERK) 1/2 was determined by immunoblotting or confocal immunolabelling. Activation of the Ca(2+)-dependent transcription factor, nuclear factor of activated T-cells (NFATc), was assessed by electromobility shift assay. RESULTS: PDGF increased the intracellular Ca2+ concentration in differentiated, but not in proliferating, PV myocytes. This is probably due to very low expression of PLC(gamma) in proliferating PV. In 6-week-old PV, PDGF stimulation induced nuclear translocation and activation of NFATc. PDGF did not induce NFATc activation in neonatal PV. PDGF-induced ERK1/2 activation was observed in both 2-4-day-old and 6-week-old PV. In 6-week-old PV, ERK1/2 activation was Ca(2+)-dependent and protein kinase C-dependent. However in 2-4-day-old PV, PDGF-induced ERK1/2 activation was via a Ca(2+)-independent, atypical protein kinase C. PLC(gamma) expression was also decreased in the neointima, compared to media, of balloon-injured rabbit subclavian arteries. CONCLUSIONS: The regulation of PDGF-induced Ca2+ increases by PLC(gamma) expression in VSM may provide a mechanism for coordinating different signaling pathways leading to activation of specific transcription factors. This may play an important role in the phenotypic modulation of VSM.

Sphingosylphosphorylcholine inhibits macrophage adhesion to vascular smooth muscle cells.

Inflammation in de-endothelialised arteries contributes to the development of cardiovascular diseases. The process that initiates this inflammatory response is the adhesion of monocytes/macrophages to exposed vascular smooth muscle cells, typically stimulated by cytokines such as tumour necrosis factor-α (TNF). The aim of this study was to determine the effect of the sphingolipid sphingosylphosphorylcholine (SPC) on the interaction of monocytes/macrophages with vascular smooth muscle cells. Rat aortic smooth muscle cells and rat bone marrow-derived macrophages were co-cultured using an in vitro assay following incubation with sphingolipids to assess inter-cellular adhesion. We reveal that SPC inhibits the TNF-induced adhesion of macrophages to smooth muscle cells. This anti-adhesive effect was the result of SPC-induced changes to the smooth muscle cells (but not the macrophages) and was mediated, at least partly, via the sphingosine 1-phosphate receptor subtype 2. Lipid raft domains were also required. Although SPC did not alter expression or membrane distribution of the adhesion proteins intercellular adhesion molecule-1 and vascular cellular adhesion protein-1 in smooth muscle cells, SPC preincubation inhibited the TNF-induced increase in inducible nitric oxide synthase (NOS2) resulting in a subsequent decrease in nitric oxide production. Inhibiting NOS2 activation in smooth muscle cells led to a decrease in the adhesion of macrophages to smooth muscle cells. This study has therefore delineated a novel pathway which can inhibit the interaction between macrophages and vascular smooth muscle cells via SPC-induced repression of NOS2 expression. This mechanism could represent a potential drug target in vascular disease.

Endothelin-1- and depolarization-induced differential regulation of cAMP response element-binding protein in proliferating and developed vascular smooth muscle.

Regulation of the transcription factor cAMP response element-binding protein (CREB) is important for gene expression in vascular smooth muscle (VSM). This study has examined the intracellular regulation of CREB by endothelin-1 (ET-1) and depolarization in native VSM, comparing proliferative and fully differentiated phenotypes. Portal veins from neonatal (proliferative) and 6-week-old (differentiated) rats were stimulated with ET-1 or K(+). In both phenotypes, CREB activation was increased by ET-1 although the time course was prolonged in neonatal VSM. This paralleled a prolonged ET-1-induced [Ca(2+)](i) increase. ET-1-induced CREB activation was dependent on extracellular signal-regulated kinase 1/2 (ERK1/2) activation and inhibited by BAPTA but not by a calmodulin-dependent protein kinase (CamK) inhibitor. In contrast, CREB activation induced by depolarization in both neonatal and developed VSM was significantly reduced by CaMK inhibition and by ERK1/2 inhibition. Therefore, CREB activation is regulated differentially in VSM depending upon stimulus; however, this is not altered in different growth states.

The cAMP-producing agonist beraprost inhibits human vascular smooth muscle cell migration via exchange protein directly activated by cAMP.

AIMS: During restenosis, vascular smooth muscle cells (VSMCs) migrate from the vascular media to the developing neointima. Preventing VSMC migration is therefore a therapeutic target for restenosis. Drugs, such as prostacyclin analogues, that increase the intracellular concentration of cyclic adenosine monophosphate (cAMP) can inhibit VSMC migration, but the mechanisms via which this occurs are unknown. Two main downstream mediators of cAMP are protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). This study has examined the effects of the prostacyclin analogue beraprost on VSMC migration and investigated the intracellular pathways involved. METHODS AND RESULTS: In a chemotaxis chamber, human saphenous vein VSMC migrated towards a platelet-derived growth-factor-BB (PDGF) chemogradient. Incubation with therapeutically relevant concentrations of cAMP-producing agonist beraprost significantly decreased PDGF-induced migration. Direct activation of either PKA or Epac inhibited migration whereas inhibition of PKA did not prevent the anti-migratory effect of beraprost. Direct activation of Epac also prevented hyperplasia in ex vivo serum-treated human veins. Using fluorescence resonance energy transfer, we demonstrated that beraprost activated Epac but not PKA. The mechanisms of this Epac-mediated effect involved activation of Rap1 with subsequent inhibition of RhoA. Cytoskeletal rearrangement at the leading edge of the cell was consequently inhibited. Interestingly, Epac1 was localized to the leading edge of migrating VSMC. CONCLUSIONS: These results indicate that therapeutically relevant concentrations of beraprost can inhibit VSMC migration via a previously unknown mechanism involving the cAMP mediator Epac. This may provide a novel target that could blunt neointimal formation.

Sphingosine 1-phosphate induces CREB activation in rat cerebral artery via a protein kinase C-mediated inhibition of voltage-gated K+ channels.

Sphingosine 1-phosphate (S1P) is a potential mitogenic stimulus for vascular smooth muscle. S1P promotes an increase in the intracellular calcium concentration ([Ca(2+)](i)) in cerebral arteries, however S1P effects on regulation of gene expression are not known. Activation of the Ca(2+)-dependent transcription factor, cAMP response element-binding protein (CREB), is associated with smooth muscle proliferation. The aim of this study was to examine the Ca(2+)-dependent mechanisms involved in S1P-induced CREB activation in cerebral artery. Western blotting and immunofluorescence with a phospho-CREB antibody were used to detect CREB activation in Sprague-Dawley rat cerebral arteries. Whole-cell patch clamp recording and single cell imaging of [Ca(2+)](i) were performed on freshly isolated cerebral artery myocytes. S1P increased activation of CREB in the nucleus of cerebral arteries. This activation was mediated by Ca(2+)/calmodulin-dependent protein kinase and was dependent on an increase in [Ca(2+)](i) via two mechanisms: (i) intracellular Ca(2+) release via an inositol 1,4,5-trisphosphate (InsP(3))-dependent pathway and (ii) Ca(2+) entry through voltage-dependent Ca(2+) channels (VDCC). Activation of the VDCC occurred through S1P-induced inhibition (approximately 50%) of the voltage-gated potassium (K(+)) current. This inhibition was via a protein kinase C-mediated pathway resulting in tyrosine phosphorylation of at least one isoform of the Kv channel (Kv 1.2). These results demonstrate that S1P can activate the transcription factor CREB through different Ca(2+)-dependent pathways including intracellular Ca(2+) release and inhibition of voltage-gated K(+) channels leading to Ca(2+) influx. Our findings suggest a potential role for S1P in regulation of gene expression in vascular smooth muscle.

Phosphoprotein enriched in astrocytes (PEA)-15: a potential therapeutic target in multiple disease states.

Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse cellular processes, such as proliferation and apoptosis, dependent upon stimulation. Regulation of these processes occurs by virtue of the unique interaction of PEA-15 with other signalling proteins. PEA-15 acts as a cytoplasmic tether for the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) preventing nuclear localisation. In order to release ERK1/2, PEA-15 requires to be phosphorylated via several potential pathways. PEA-15 (and its phosphorylation state) therefore regulates many ERK1/2-dependent processes, including proliferation, via regulating ERK1/2 nuclear translocation. In addition, PEA-15 contains a death effector domain (DED) which allows interaction with other DED-containing proteins. PEA-15 can bind the DED-containing apoptotic adaptor molecule, Fas-associated death domain protein (FADD) which is also dependent on the phosphorylation status of PEA-15. PEA-15 binding of FADD can inhibit apoptosis as bound FADD cannot participate in the assembly of apoptotic signalling complexes. Through these protein-protein interactions, PEA-15-regulated cellular effects have now been investigated in a number of disease-related studies. Changes in PEA-15 expression and regulation have been observed in diabetes mellitus, cancer, neurological disorders and the cardiovascular system. These changes have been suggested to contribute to the pathology related to each of these disease states. As such, new therapeutic targets based around PEA-15 and its associated interactions are now being uncovered and could provide novel avenues for treatment strategies in multiple diseases.

Long-term zinc deprivation accelerates rat vascular smooth muscle cell proliferation involving the down-regulation of JNK1/2 expression in MAPK signaling.

BACKGROUND: The accelerated proliferation of vascular smooth muscle cells (VSMCs) is a contributor for atherosclerosis by thickening the vascular wall. Since zinc modulation of VSMC proliferation has not been clarified, this study investigated whether zinc affects VSMC proliferation. METHODS AND RESULTS: Both a rat aorta origin vascular smooth muscle cell line (A7r5 VSMCs) and primary VSMCs which were collected from rat aorta (pVSMCs) were cultured with zinc (0-50 µM Zn) for short- (≤12 d) and long-term (28 d) periods under normal non-calcifying (0 or 1 mM P) or calcifying (>2 mM P) P conditions. Mouse vascular endothelial cells (MS I cells) were also cultured (under 0-50 µM Zn and 10 mM P for 20 d) to compare with VSMC cultures. While during short-term culture of VSMCs, zinc deprivation decreased cell proliferation in a zinc-concentration manner both under non-calcifying and calcifying conditions in A7r5 and pVSMCs (P < 0.05), during long-term cultures (28 d), A7r5 VSMC proliferation was inversely related to medium zinc concentration under normal physiological P conditions (regression coefficient r(2) = -0.563, P = 0.012). The anti-cell proliferative effect of zinc supplementation (>50 µM) was VSMC-specific. Long-term (35 d), low zinc treatment down-regulated JNK expression and activation, while not affecting ERK1/2 MAPK signaling in A7r5 VSMCs. CONCLUSION: The results showed that chronic zinc deprivation accelerated VSMC proliferation, perhaps due to down-regulation of MAPK-JNK signaling, and that the anti-cell proliferative role of zinc is VSMC-specific. The findings suggested that zinc may have anti-VSMC proliferative properties in atherosclerosis.

Marginal dietary zinc deficiency in vivo induces vascular smooth muscle cell apoptosis in large arteries.

AIMS: Dietary zinc deficiency has been associated with the development of atherosclerosis although the effects on vascular smooth muscle cells (VSMCs), important in maintaining atherosclerotic plaque integrity, are unknown. The main aim of this study was to elucidate the effect of a zinc-deficient environment on VSMCs using an in vivo model. METHODS AND RESULTS: Rats were maintained for 2 weeks on a marginally zinc-deficient diet which resulted in a significant reduction in plasma zinc levels. Large arteries from zinc-deficient rats had significantly increased apoptosis within the VSMC layers compared with arteries from rats on a zinc-adequate diet. This apoptosis occurred in parallel with a known apoptotic pathway, namely dephosphorylation of the pro-apoptotic protein Bcl-2-associated death promoter protein (BAD). Activation of extracellular signal-regulated kinase (ERK)1/2, which maintains BAD phosphorylation as a pro-survival mechanism, was decreased in arteries from zinc-deficient rats. The mechanisms of this in vivo effect were investigated in vitro. Cultured rat VSMCs incubated with plasma from zinc-deficient rats similarly resulted in increased apoptosis in parallel with BAD dephosphorylation and decreased ERK1/2 activation. Further related apoptotic mechanisms induced by plasma from zinc-deficient rats involved a prolonged rise in [Ca²âº]i leading to subsequent activation of the phosphatase calcineurin. Calcineurin activation was required to dephosphorylate BAD. In addition, an increase in oxidative stress contributed to the apoptotic effect induced by plasma from zinc-deficient rats. CONCLUSION: In conclusion, a marginally zinc-deficient diet is pro-apoptotic for VSMCs and this may contribute to cardiovascular disease.

Suboptimal dietary zinc intake promotes vascular inflammation and atherogenesis in a mouse model of atherosclerosis.

SCOPE: Cardiovascular health is strongly influenced by diet. Zinc has antioxidant and anti-inflammatory properties but its long-term influence on vascular health at dietary intake levels relevant to the human population in developed countries has not been studied. We investigated the influence of suboptimal zinc intake in a Western-type diet on the development of vascular inflammation and arterial plaque in apoE knock-out (AEKO) mice. METHODS AND RESULTS: Weanling AEKO and wild-type (WT) controls were given high saturated fat (21% w/w) and high cholesterol (0.15%) semi-synthetic diets containing 3 or 35 mg Zn/kg (AEKO and WT) or 8 mg Zn/kg (AEKO only) for over 6 months. AEKO mice on zinc intakes of 3 and 8 mg Zn/kg (suboptimal zinc) developed significantly (p < 0.05) more aortic plaque than AEKO mice consuming 35 mg Zn/kg (adequate zinc). Circulating levels of interleukin-1ß, interleukin-6 and soluble vascular adhesion molecule-1 were significantly (p < 0.05) raised at the lowest zinc intake in AEKO mice, as compared to zinc-adequate controls. Plasma total cholesterol and total protein were also significantly (p < 0.05) increased at the lowest zinc intake. CONCLUSION: We propose that suboptimal dietary zinc intake raises circulating pro-atherogenic lipoprotein levels that promote vascular inflammation and enhance arterial plaque formation.