Increased microvascular permeability in mice lacking Epac1 (Rapgef3).

AIM: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. METHODS: Epac1-/- and Epac2-/- C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. RESULTS: Epac1-/- mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1-/- mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2-/- and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1-/- microvascular interendothelial junctions had constitutively less junctional complex. CONCLUSION: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity.

Drug repurposing: sulfasalazine sensitizes gliomas to gamma knife radiosurgery by blocking cystine uptake through system Xc-, leading to glutathione depletion.

Glioblastomas (GBMs) are aggressive brain tumors that always recur after radiotherapy. Cystine, mainly provided by the system X(c)(-) antiporter, is a requirement for glioma cell synthesis of glutathione (GSH) which has a critical role in scavenging free radicals, for example, after radiotherapy. Thus, we hypothesized that the X(c)(-)-inhibitor sulfasalazine (SAS) could potentiate the efficacy of radiotherapy against gliomas. Here, we show that the catalytic subunit of system X(c)(-), xCT, was uniformly expressed in a panel of 30 human GBM biopsies. SAS treatment significantly reduced cystine uptake and GSH levels, whereas it significantly increased the levels of reactive oxygen species (ROS) in glioma cells in vitro. Furthermore, SAS and radiation synergistically increased DNA double-strand breaks and increased glioma cell death, whereas adding the antioxidant N-acetyl-L-cysteine (NAC) reversed cell death. Moreover, SAS and gamma knife radiosurgery (GKRS) synergistically prolonged survival in nude rats harboring human GBM xenografts, compared with controls or either treatment alone. In conclusion, SAS effectively blocks cystine uptake in glioma cells in vitro, leading to GSH depletion and increased ROS levels, DNA damage and cell death. Moreover, it potentiates the anti-tumor efficacy of GKRS in rats with human GBM xenografts, providing a survival benefit. Thus, SAS may have a role as a radiosensitizer to enhance the efficacy of current radiotherapies for glioma patients.

The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling.

Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for this phenomenon. The progenitor cell marker NG2/melanoma proteoglycan (MPG) is aberrantly expressed by various tumors, but its role in cell death signaling and its potential as a therapeutic target are largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma spheroids from 15 patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and tumor necrosis factor (TNF)alpha. High NG2/MPG expression correlated with multidrug resistance mediated by increased activation of alpha3beta1 integrin/PI3K signaling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated beta1 integrin signaling revealing potent antitumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the antiapoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

Proteomic and computational methods in systems modeling of cellular signaling.

Cellular signaling lies at the core of cellular behavior, and is central for the understanding of many pathologic conditions. To comprehend how signal transduction is orchestrated at the molecular level remains the ultimate challenge for cell biology. In the last years there has been a revolution in the development of high-throughput methodologies in proteomics and genomics, which have provided extensive knowledge about expression profiles and molecular interaction-networks. However, these methods have typically provided qualitative and static information. This is about to turn, and several high-throughput methods are now available that provide quantitative and temporal information. These data are well suited for analysis by computational methods and bioinformatics, which are becoming increasingly valuable tools to grasp the complexity of cellular networks. At present, several cellular pathways have been modeled in silico and the analysis provides new understanding of the underlying properties that contribute to their dynamic features. Here, we review methodologies that are used for in silico modeling as well as methods to obtain large-scale quantitative data, and discuss how they can be integrated to generate powerful and predictive models of cellular processes. We argue that the generation of such models provide powerful tools to understand how systems properties emerges in healthy and pathologic states, and to generate efficient strategies for pharmacological intervention.

Adrenaline potentiates PI 3-kinase in platelets stimulated with thrombin and SFRLLN: role of secreted ADP.

Adrenaline significantly potentiated late thrombin- and SFRLLN-induced PtdIns(3,4)P(2) production. Furthermore, the potentiating effect of adrenaline on thrombin-induced PtdIns(3, 4)P(2) production was independent on secreted ADP, whereas, the effect of adrenaline on SFRLLN-induced PtdIns(3,4)P(2) production was completely dependent of secreted ADP. However, the ADP-dependent accumulation of PtdIns(3,4)P(2) was not required for irreversible platelet aggregation induced by SFRLLN in the presence of adrenaline. It is concluded that adrenaline can replace secreted ADP to potentiate PtdIns(3,4)P(2) production in thrombin-stimulated but not in SFRLLN-stimulated platelets, thus demonstrating a qualitative difference between platelet stimulation by thrombin and the thrombin receptor activating peptide SFRLLN.

PI 3-kinase signalling in platelets: the significance of synergistic, autocrine stimulation.

Phosphoinositide 3-kinases (PI 3Ks) play a key role in regulation of intracellular signalling and cellular function, including cell proliferation, apoptosis, chemotaxis, membrane trafficking and platelet activation. The PI 3Ks are grouped into three classes on the basis on their structure and in vitro substrate specificity. Class I are activated by a variety of agonists which mediate their effect through tyrosine kinase-linked or G-protein-linked receptors. In vivo class I PI 3Ks seem to preferentially phosphorylate the D3 hydroxyls of the inositol moiety of PtdIns(4,5)P2 to produce PtdIns(3,4,5)P3. However, class II PI 3Ks preferentially phosphorylate the D3 hydroxyl of PtdIns and PtdIns(4)P to produce PtdIns(3)P and PtdIns(3,4)P2, respectively. The late accumulation of PtdIns(3,4)P2 has been suggested to play an important role in irreversible platelet aggregation. In human platelets the class II PI 3K isoform HsC2-PI 3K is activated in an integrin alpha IIb beta 3 + fibrinogen-dependent manner. Class III PI 3Ks phosphorylate PtdIns to produce PtdIns(3)P, which play a crucial role in vesicular trafficking. Recent work has suggested that crosstalk between individual receptors and their downstream signal pathways play a central role in PI 3K signalling responses. In this review, we will concentrate on recent advances regarding the regulation of platelet PI 3Ks.

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Formation of PI 3-kinase products in platelets by thrombin, but not collagen, is dependent on synergistic autocrine stimulation, particularly through secreted ADP.

Platelet activation by thrombin or collagen results in secretion and synthesis of several platelet agonists that enhance the responses to the primary agonists (autocrine stimulation). To disclose the effects of thrombin and collagen on the phosphorylation of 3-phosphoinositides per se we incubated platelets with five inhibitors of platelet autocrine stimulation (IAS) that act extracellularly. We found that IAS almost totally blocked thrombin-induced production of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]. In contrast, collagen induced massive production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) in the presence of IAS. When testing the effect of each inhibitor individually we found the strongest inhibition of thrombin-induced PtdIns(3,4)P(2) production with the ADP scavenger system CP/CPK. Furthermore, we found a strong synergistic effect between exogenously added ADP and thrombin on production of PtdIns(3,4)P(2). In contrast to the results from 3-phosphorylated phosphoinositides, CP/CPK had little effect on thrombin-induced protein tyrosine phosphorylation. Our results show the importance of autocrine stimulation in thrombin-induced accumulation of 3-phosphorylated phosphoinositides and raise the question as to whether thrombin by itself is capable of inducing PI 3-K activation. In marked contrast to thrombin, collagen per se appears to be able to trigger increased production of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3).

Platelet-derived growth factor inhibits platelet activation in heparinized whole blood.

We previously have demonstrated that human platelets have functionally active platelet-derived growth factor alpha-receptors. Studies with gel-filtered platelets showed that an autocrine inhibition pathway is transduced through this tyrosine kinase receptor during platelet activation. The physiological significance of this inhibitory effect of platelet-derived growth factor on gel-filtered platelets activation is, however, not known. In the present study, we investigated whether platelet-derived growth factor inhibits platelet activation under more physiological conditions in heparinized whole blood, which represents a more physiological condition than gel-filtered platelets. Using flow cytometric assays, we demonstrate here that platelet-derived growth factor inhibits thrombin-, thrombin receptor agonist peptide SFLLRN-, and collagen-induced platelet aggregation and shedding of platelet-derived microparticles from the platelet plasma membrane during platelet aggregation in stirred heparinized whole blood. The inhibitory effect of platelet-derived growth factor was dose dependent. However, under nonaggregating conditions (no stirring), we could not demonstrate any significant effect of platelet-derived growth factor on thrombin- and thrombin receptor agonist peptide-induced platelet surface expression of P-selectin. Our results demonstrate that platelet-derived growth factor appears to be a true antithrombotic agent only under aggregating conditions in heparinized whole blood.