PTEN inhibits AMPK to control collective migration

PTEN is one of the most frequently mutated tumor suppressor gene in cancer. PTEN is generally altered in invasive cancers such as glioblastomas, but its function in collective cell migration and invasion is not fully characterized. Herein, we report that the loss of PTEN increases cell speed during collective migration of non-tumourous cells both in vitro and in vivo. We further show that loss of PTEN promotes LKB1-dependent phosphorylation and activation of the major metabolic regulator AMPK. In turn AMPK increases VASP phosphorylation, reduces VASP localization at cell-cell junctions and decreases the interjunctional transverse actin arcs at the leading front, provoking a weakening of cell-cell contacts and increasing migration speed. Targeting AMPK activity not only slows down PTEN-depleted cells, it also limits PTEN-null glioblastoma cell invasion, opening new opportunities to treat glioblastoma lethal invasiveness.

Naringenin Inhibits Platelet Activation and Arterial Thrombosis Through Inhibition of Phosphoinositide 3-Kinase and Cyclic Nucleotide Signaling

Citrus flavanoids intake can reduce the risk of cardiovascular diseases. Naringenin, a natural predominant flavonoid abundant in citrus fruits, possesses protective effects against atherothrombotic diseases. As platelet activation plays central roles in atherothrombogenesis, we studied the effects of naringenin on platelet activation, signaling, thrombosis and hemostasis. Naringenin dose-dependently inhibited agonist-induced platelet aggregation in vitro, and exhibited more-potent efficacy on ADP-induced platelet aggregation. It also suppressed platelet aggregation stimulated by ADP ex vivo. Naringenin inhibited ADP-induced platelet α-granule secretion, fibrinogen binding, intracellular calcium mobilization and platelet adhesion on collagen-coated surface. Naringenin also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in integrin signaling. Mechanism studies indicated that naringenin suppressed PI3K-mediated signaling and phosphodiesterase activity in platelets, in addition to increasing cGMP levels and VASP phosphorylation at Ser239. Furthermore, naringenin-induced VASP phosphorylation and inhibition of platelet aggregation were reversed by a PKA inhibitor treatment. Interestingly, naringenin inhibited thrombus formation in the (FeCl3)-induced rat carotid arterial thrombus model, but not cause a prolonged bleeding time in mice. This study suggests that naringenin may represent a potential antiplatelet agent targeting PI3K and cyclic nucleotide signaling, with a low bleeding risk.

cGMP signaling: probing antagonistic cyclic nucleotide platelet signals by modeling and experiment

BackgroundThe cyclic nucleotides cAMP and cGMP inhibit platelet activation.ResultsWe extended an older model and systematically integrated drugs as external stimuli. Data driven modeling allowed us to design models that provide a quantitative output for quantitative input information. This relies on condensed information about involved regulation and modeling of pharmacological interventions by systematic optimization methods. By multi-experiment fitting, we validated our model optimizing the parameters of the model. In addition, we show how the output of the developed cGMP model can be used as input for a modular model of VASP phosphorylation and for the activity of cAMP and cGMP pathways in platelets.ConclusionsWe present a model for cGMP signaling and VASP phosphorylation, that allows to estimate drug action on any of the inhibitory cyclic nucleotide pathways (cGMP, cAMP) and has been validated by experimental data.

The PKGIα/VASP pathway is involved in insulin- and high glucose-dependent regulation of albumin permeability in cultured rat podocytes

Podocytes, the principal component of the glomerular filtration barrier, regulate glomerular permeability to albumin via their contractile properties. Both insulin- and high glucose (HG)-dependent activation of protein kinase G type Iα (PKGIα) cause reorganization of the actin cytoskeleton and podocyte disruption. Vasodilator-stimulated phosphoprotein (VASP) is a substrate for PKGIα and involved in the regulation of actin cytoskeleton dynamics. We investigated the role of the PKGIα/VASP pathway in the regulation of podocyte permeability to albumin. We evaluated changes in high insulin- and/or HG-induced transepithelial albumin flux in cultured rat podocyte monolayers. Expression of PKGIα and downstream proteins was confirmed by western blot and immunofluorescence. We demonstrate that insulin and HG induce changes in the podocyte contractile apparatus via PKGIα-dependent regulation of the VASP phosphorylation state, increase VASP colocalization with PKGIα, and alter the subcellular localization of these proteins in podocytes. Moreover, VASP was implicated in the insulin- and HG-dependent dynamic remodelling of the actin cytoskeleton and, consequently, increased podocyte permeability to albumin under hyperinsulinaemic and hyperglycaemic conditions. These results indicate that insulin- and HG-dependent regulation of albumin permeability is mediated by the PKGIα/VASP pathway in cultured rat podocytes. This molecular mechanism may explain podocytopathy and albuminuria in diabetes.

Head-to-Head Comparison of Consensus-Recommended Platelet Function Tests to Assess P2Y12 Inhibition—Insights for Multi-Center Trials

The vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by Multiplate® whole-blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR-C 66096). Platelet function testing was carried out simultaneously by Multiplate® aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA- and flow cytometry-based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with Multiplate® was slightly higher for the flow cytometry-based VASP assay (r = 0.79, p < 0.0001) than for the ELISA-based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (κ between 0.86 and 0.92). In conclusion, the consensus-recommended assays with their standardized cut-offs should not be used interchangeably in multi-center clinical studies but, rather, they should be standardized throughout sites.

Hypoxia Modulates Platelet Purinergic Signalling Pathways

Background Hypoxia resulting from ascent to high-altitude or pathological states at sea level is known to increase platelet reactivity. Previous work from our group has suggested that this may be adenosine diphosphate (ADP)-specific. Given the clinical importance of drugs targeting ADP pathways, research into the impact of hypoxia on platelet ADP pathways is highly important. Methods Optimul aggregometry was performed on plasma from 29 lowland residents ascending to 4,700 m, allowing systematic assessment of platelet reactivity in response to several platelet agonists. Aggregometry was also performed in response to ADP in the presence of inhibitors of the two main ADP receptors, P2Y1 and P2Y12 (MRS2500 and cangrelor, respectively). Phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key determinant of platelet aggregation, was analysed using the VASPFix assay. Results Hypobaric hypoxia significantly reduced the ability of a fixed concentration of cangrelor to inhibit ADP-induced aggregation and increased basal VASP phosphorylation. However, in the absence of P2Y receptor inhibitors, we did not find evidence of increased platelet sensitivity to any of the agonists tested and found reduced sensitivity to thrombin receptor-activating peptide-6 amide. Conclusion Our results provide evidence of increased P2Y1 receptor activity at high altitude and suggest down-regulation of the P2Y12 pathway through increased VASP phosphorylation. These changes in ADP pathway activity are of potential therapeutic significance to high-altitude sojourners and hypoxic sea level patients prescribed platelet inhibitors and warrant further investigation.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

Natural Coumarin Derivative Esculetin Regulates Platelet Activation via Modulating NF-κB Signaling in Cyclic Nucleotide-Independent Manner

Esculetin, a natural coumarin derivative, shows exciting biological activities in a variety of cell and animal models. Our recent study demonstrated that esculetin exhibits antiplatelet effects by obstructing the phospholipase C γ2/protein kinase C cascade, hydroxyl radical formation, and Akt activation. In this study, we further examined the involvement of cyclic 3′-5′adenosine monophosphate/, vasodilator-stimulated phosphoprotein (VASP), integrin αIIbβ3, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), since cyclic nucleotides reduce the phosphorylation of VASP and activate NF-κB, subsequently inducing αIIbβ3 activation that significantly involves the platelet inhibitory pathways. We found that esculetin (50 and 80 µM) did not significantly affect fibrinogen-induced aggregation of elastase-treated platelets; however, it markedly blocked integrin αIIbβ3 activation by interrupting the binding of fluorescein isothiocyanate-labeled PAC-1. In addition, neither ODQ nor SQ22536 significantly reversed esculetin-mediated antiplatelet activity stimulated by collagen. Nitroglycerin and prostaglandin E1 significantly increased VASP phosphorylation, but esculetin had no effect in this reaction, the values being almost identical with those of normal platelets. Furthermore, esculetin, at its maximum concentration of 80 μM significantly reduced the phosphorylation of IκBα and p65 and reversed IκBα degradation in collagen-induced platelets. These results suggest that the NF-κB-dependent αIIbβ3 inhibition of esculetin might represent a novel feedback inhibitory mechanism to regulate platelet functions.