Differential effects of single fatty acids and fatty acid mixtures on the phosphoinositide 3-kinase/Akt/eNOS pathway in endothelial cells.

SCOPE: Dietary fat composition is an important modulator of vascular function. Non-esterified fatty acids (NEFA) enriched in saturated fatty acids (SFA) are thought to reduce vascular reactivity by attenuating insulin signalling via vasodilator pathways (phosphoinositide 3-kinase (PI3K)/Akt/endothelial nitric oxide synthase (eNOS)) and enhancing signalling via pro-inflammatory pathways. METHODS: To examine the effects of fatty acids on these pathways, human aortic endothelial cells were incubated with single fatty acids, and mixtures of these fatty acids to mimic typical NEFA composition and concentrations achieved in our previous human study. RNA was extracted to determine gene expression using real-time RT-PCR and cell lysates prepared to assess protein phosphorylation by Western blotting. RESULTS: Oleic acid (OA, 100 µM) was shown to down regulate expression of the insulin receptor, PTEN and a PI3K catalytic (p110ß) and regulatory (p85α) subunit compared to palmitic, linoleic and stearic acids (P < 0.04), and promote greater eNOS phosphorylation at Ser1177. Both concentration and composition of the SFA and SFA plus n-3 polyunsaturated fatty acids (PUFA) mixtures had significant effects on genes involved in the PI3K/Akt pathway. Greater up-regulation was found with 800 than 400 µM concentration (respective of concentrations in insulin resistant and normal individuals), whereas greater down-regulation was evident with SFA plus n-3 PUFA than SFA mixture alone. CONCLUSION: Our findings provide novel insights into the modulation of the PI3K/Akt/eNOS pathway by single fatty acids and fatty acid mixtures. In particular, OA appears to promote signalling via this pathway, with further work required to determine the primary molecular site(s) of action.

Regulation of Early Steps of GPVI Signal Transduction by Phosphatases: A Systems Biology Approach.

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.

Ibrutinib Inhibits Platelet Integrin αIIbß3 Outside-In Signaling and Thrombus Stability But Not Adhesion to Collagen.

OBJECTIVE: Ibrutinib is an irreversible Bruton tyrosine kinase inhibitor approved for treatment of Waldenstrom macroglobulinemia, chronic lymphocytic leukemia, and mantle cell lymphoma that increases the risk of bleeding among patients. Platelets from ibrutinib-treated patients exhibit deficiencies in collagen-evoked signaling in suspension; however, the significance of this observation and how it relates to bleeding risk is unclear, as platelets encounter immobile collagen in vivo. We sought to clarify the effects of ibrutinib on platelet function to better understand the mechanism underlying bleeding risk. APPROACH AND RESULTS: By comparing signaling in suspension and during adhesion to immobilized ligands, we found that the collagen signaling deficiency caused by ibrutinib is milder during adhesion to immobilized collagen. We also found that platelets in whole blood treated with ibrutinib adhered to collagen under arterial shear but formed unstable thrombi, suggesting that the collagen signaling deficiency caused by ibrutinib may not be the predominant cause of bleeding in vivo. However, clot retraction and signaling evoked by platelet adhesion to immobilized fibrinogen were also inhibited by ibrutinib, indicating that integrin αIIbß3 outside-in signaling is also effected in addition to GPVI signaling. When ibrutinib was combined with the P2Y12 inhibitor, cangrelor, thrombus formation under arterial shear was inhibited additively. CONCLUSIONS: These findings suggest that (1) ibrutinib causes GPVI and integrin αIIbß3 platelet signaling deficiencies that result in formation of unstable thrombi and may contribute toward bleeding observed in vivo and (2) combining ibrutinib with P2Y12 antagonists, which also inhibit thrombus stability, may have a detrimental effect on hemostasis.

A model to create an efficient and equitable admission policy for patients arriving to the cardiothoracic ICU.

OBJECTIVE: To develop queuing and simulation-based models to understand the relationship between ICU bed availability and operating room schedule to maximize the use of critical care resources and minimize case cancellation while providing equity to patients and surgeons. DESIGN: Retrospective analysis of 6-month unit admission data from a cohort of cardiothoracic surgical patients, to create queuing and simulation-based models of ICU bed flow. Three different admission policies (current admission policy, shortest-processing-time policy, and a dynamic policy) were then analyzed using simulation models, representing 10 yr worth of potential admissions. Important output data consisted of the "average waiting time," a proxy for unit efficiency, and the "maximum waiting time," a surrogate for patient equity. SETTING: A cardiothoracic surgical ICU in a tertiary center in New York, NY. PATIENTS: Six hundred thirty consecutive cardiothoracic surgical patients admitted to the cardiothoracic surgical ICU. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Although the shortest-processing-time admission policy performs best in terms of unit efficiency (0.4612 days), it did so at expense of patient equity prolonging surgical waiting time by as much as 21 days. The current policy gives the greatest equity but causes inefficiency in unit bed-flow (0.5033 days). The dynamic policy performs at a level (0.4997 days) 8.3% below that of the shortest-processing-time in average waiting time; however, it balances this with greater patient equity (maximum waiting time could be shortened by 4 days compared to the current policy). CONCLUSIONS: Queuing theory and computer simulation can be used to model case flow through a cardiothoracic operating room and ICU. A dynamic admission policy that looks at current waiting time and expected ICU length of stay allows for increased equity between patients with only minimum losses of efficiency. This dynamic admission policy would seem to be a superior in maximizing case-flow. These results may be generalized to other surgical ICUs.

Scenario-robust pre-disaster planning for multiple relief items.

The increasing vulnerability of the population from frequent disasters requires quick and effective responses to provide the required relief through effective humanitarian supply chain distribution networks. We develop scenario-robust optimization models for stocking multiple disaster relief items at strategic facility locations for disaster response. Our models improve the robustness of solutions by easing the difficult, and usually impossible, task of providing exact probability distributions for uncertain parameters in a stochastic programming model. Our models allow decision makers to specify uncertainty parameters (i.e., point and probability estimates) based on their degrees of knowledge, using distribution-free uncertainty sets in the form of ranges. The applicability of our generalized approach is illustrated via a case study of hurricane preparedness in the Southeastern United States. In addition, we conduct simulation studies to show the effectiveness of our approach when conditions deviate from the model assumptions.

Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration.

The lipid products of phosphoinositide 3-kinase (PI3K) are involved in many cellular responses such as proliferation, migration, and survival. Disregulation of PI3K-activated pathways is implicated in different diseases including cancer and diabetes. Among the three classes of PI3Ks, class I is the best characterized, whereas class II has received increasing attention only recently and the precise role of these isoforms is unclear. Similarly, the role of phosphatidylinositol-3-phosphate (PtdIns-3-P) as an intracellular second messenger is only just beginning to be appreciated. Here, we show that lysophosphatidic acid (LPA) stimulates the production of PtdIns-3-P through activation of a class II PI3K (PI3K-C2beta). Both PtdIns-3-P and PI3K-C2beta are involved in LPA-mediated cell migration. This study is the first identification of PtdIns-3-P and PI3K-C2beta as downstream effectors in LPA signaling and demonstration of an intracellular role for a class II PI3K. Defining this novel PI3K-C2beta-PtdIns-3-P signaling pathway may help clarify the process of cell migration and may shed new light on PI3K-mediated intracellular events.

A model of the PI cycle reveals the regulating roles of lipid-binding proteins and pitfalls of using mosaic biological data.

The phosphatidylinositol (PI) cycle is central to eukaryotic cell signaling. Its complexity, due to the number of reactions and lipid and inositol phosphate intermediates involved makes it difficult to analyze experimentally. Computational modelling approaches are seen as a way forward to elucidate complex biological regulatory mechanisms when this cannot be achieved solely through experimental approaches. Whilst mathematical modelling is well established in informing biological systems, many models are often informed by data sourced from multiple unrelated cell types (mosaic data) or from purified enzyme data. In this work, we develop a model of the PI cycle informed by experimental and omics data taken from a single cell type, namely platelets. We were able to make a number of predictions regarding the regulation of PI cycle enzymes, the importance of the number of receptors required for successful GPCR signaling and the importance of lipid- and protein-binding proteins in regulating second messenger outputs. We then consider how pathway behavior differs, when fully informed by data for HeLa cells and show that model predictions remain consistent. However, when informed by mosaic experimental data model predictions greatly vary illustrating the risks of using mosaic datasets from unrelated cell types.

Phosphoinositide (PI) 3-kinase assays.

The regulation of phosphoinositide (PI) 3-kinase activities has been linked to many normal and disease-related processes, including cell survival, cell growth and proliferation, cell differentiation, cell motility, and intracellular vesicle trafficking. However, as the family of enzymes has now grown to include eight true members, in three functional classes, plus several related protein kinases that are also inhibited by the widely used PI 3-kinase selective inhibitors, wortmannin and LY294002, extended methodologies are required to identify which type of kinase is involved in a particular cellular process, or protein complex, under study. A robust in vitro PI 3-kinase assay, suitable for use with immunoprecipitates, or purified proteins, is described here together with a series of modifications of substrate and assay conditions that will aid researchers in the identification of the particular class and isoform of PI 3-kinase that is involved in a signaling process under investigation.

Precise Quantification of Platelet Proteins and Their Phosphorylation States.

Systems biology and modeling approaches require quantitative data-rich temporal experiments to better understand the dynamics and regulation of the components of the signaling pathways that governs cell biology and physiology. Here we present a modified Western blotting method to rapidly analyze and accurately quantify protein copy number, and their respective phosphorylation states at specific sites over detailed time courses.

Regulation of glycogen synthase kinase 3 in human platelets: a possible role in platelet function?

In this study we show that both glycogen synthase kinase 3 (GSK3) isoforms, GSK3alpha and GSK3beta, are present in human platelets and are phosphorylated on Ser(21) and Ser(9), respectively, in platelets stimulated with collagen, convulxin and thrombin. Phosphorylation of GSK3alpha/beta was dependent on phosphoinositide 3-kinase (PI3K) activity and independent of platelet aggregation, and correlated with a decrease in GSK3 activity that was preserved by pre-incubating platelets with PI3K inhibitor LY294002. Three structurally distinct GSK3 inhibitors, lithium, SB415286 and TDZD-8, were found to inhibit platelet aggregation. This implicates GSK3 as a potential regulator of platelet function.

Enhanced sensitivity to rapamycin following long-term oestrogen deprivation in MCF-7, T-47-D and ZR-75-1 human breast cancer cells.

Human breast cancer cells (MCF-7, T-47-D and ZR-75-1) can adapt to circumvent any reduced growth rate during long-term oestrogen deprivation, and this provides three model systems to investigate mechanisms of endocrine resistance in breast cancer. In this paper we report consistent differences in the effects of three growth inhibitors following long-term oestrogen deprivation in all three cell models. Long-term oestrogen deprivation of MCF-7, T-47-D and ZR-75-1 cells resulted in reduced growth inhibition by PD98059 (2-10 µg/ml), implying a loss of dependence on mitogen-activated protein kinase pathways for growth. The growth inhibitor LY294002 (2-10 µM) inhibited growth of both oestrogen-maintained and oestrogen-deprived cells with similar dose-responses, implying continued similar dependence on phosphoinositide 3-kinase (PI3K) pathways with no alteration after adaptation to oestrogen independent growth. However, by contrast, long-term oestrogen deprivation resulted in an increased sensitivity to growth inhibition by rapamycin, which was not reduced by readdition of oestradiol. The enhanced inhibition of long-term oestrogen-deprived MCF-7-ED, T-47-D-ED and ZR-75-1-ED cell growth by combining rapamycin with LY294002 at concentrations where each alone had little effect, offers preclinical support to the development of therapeutic combinations of rapamycin analogues with other PI3K inhibitors in endocrine-resistant breast cancer.

Are class II phosphoinositide 3-kinases potential targets for anticancer therapies?

Of the three classes of true phosphoinositide (PI) 3-kinases, the class II subdivision, which consists of three isoforms, PI3K-C2alpha, PI3K-C2beta and PI3K-C2gamma, is the least well understood. There are a number of reasons for this. This class of PI 3-kinase was identified exclusively by PCR and homology cloning approaches and not on the basis of cellular function. Like class I PI 3-kinases, class II PI 3-kinases are activated by diverse receptor types. To complicate the elucidation of class II PI 3-kinase function further, their in vitro substrate specificity is intermediate between the receptor activated class I PI 3-kinases and the housekeeping class III PI 3-kinase. The class II PI 3-kinases are inhibited by the two commonly used PI 3-kinase family selective inhibitors, wortmannin and LY294002, and there are no widely available, specific inhibitors for the individual classes or isoforms. Here the current state of understanding of class II PI 3-kinase function is reviewed, followed by an appraisal as to whether there is enough evidence to suggest that pharmaceutical companies, who are currently targeting the class I PI 3-kinases in an attempt to generate anticancer agents, should also consider targeting the class II PI 3-kinases.

PtdIns5P activates the host cell PI3-kinase/Akt pathway during Shigella flexneri infection.

The virulence factor IpgD, delivered into nonphagocytic cells by the type III secretion system of the pathogen Shigella flexneri, is a phosphoinositide 4-phosphatase generating phosphatidylinositol 5 monophosphate (PtdIns5P). We show that PtdIns5P is rapidly produced and concentrated at the entry foci of the bacteria, where it colocalises with phosphorylated Akt during the first steps of infection. Moreover, S. flexneri-induced phosphorylation of host cell Akt and its targets specifically requires IpgD. Ectopic expression of IpgD in various cell types, but not of its inactive mutant, or addition of short-chain penetrating PtdIns5P is sufficient to induce Akt phosphorylation. Conversely, sequestration of PtdIns5P or reduction of its level strongly decreases Akt phosphorylation in infected cells or in IpgD-expressing cells. Accordingly, IpgD and PtdIns5P production specifically activates a class IA PI 3-kinase via a mechanism involving tyrosine phosphorylations. Thus, S. flexneri parasitism is shedding light onto a new mechanism of PI 3-kinase/Akt activation via PtdIns5P production that plays an important role in host cell responses such as survival.