Abnormal platelet amyloid-ß precursor protein metabolism in SAMP8 mice: Evidence for peripheral marker in Alzheimer's disease.

Senescence-accelerated mouse strains have proved to be an accelerated-aging model, which mimics numerous features with Alzheimer's disease (AD). Three, six, and nine-month senescence-accelerated resistant 1 and senescence-accelerated prone 8 (SAMP8) mice were used in the current study, to unravel potential mechanisms for dementia and explore new diagnostic approaches for AD. The amyloid-ß (Aß40) and Aß42 levels were elevated in hippocampi and platelets from SAMP8, along with a reduced α-secretase expression and an enhanced ß-secretase expression extent with age, compared to control mice. Furthermore, hippocampal Aß40 and Aß42 of SAMP8 were positively correlated with platelet of these mice with aging progression. In addition, ß-γ-secretase-modulated proteolytic proceeding of amyloid precursor protein in platelet might work through the PI3K/Akt/GSK3ß pathway. These results indicate that platelet could be a potential early marker in the periphery to study the age-correlative aggregation of the amyloid-ß peptide in patients with AD, while still requiring the considerable study.

PI3K pathway and Bcl-2 family. Clinicopathological features in prostate cancer.

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathways and Bcl-2 family play a central role in prostate cancer (PC). The aim was to determine influence in the biochemical progression in PC. To evaluate the association between clinic pathological and immunohistochemical variables, Spearman's test was performed. Log-rank test and Kaplan-Meier curves were used for survival comparisons. To explore the correlation of the studied immunohistochemical parameters and the established prognostic variables with biochemical progression, univariate and multivariate Cox proportional Hazard regression analyses were performed. Spearman analysis showed correlation between stroma expression and tumor expression of PI3K with biochemical progression (p = .009, p = .004), respectively, and tumor immunohistochemical score with biochemical progression (p = .051). In the multivariate Cox regression model, only PI3K was retained as independent predictors of biochemical progression. In stroma expression, PI3K is (HR 0.172, 95% CI 0.065-0.452, p = .000); tumor expression, PI3K is (HR 0.087, 95% CI 0.026-0.293, p = .000), and tumor immunohistochemical score (HR 0.382, 95% CI 0.209-0.697 p = .002). Our results suggest a role for prostatic expression of PI3K was prognostic markers for PC. PI3K/AKT/mTOR and Bcl-2 family are becoming an important therapeutic target and predictive biomarkers of onset and progression of PC.

A phase 1 trial of temsirolimus and intensive re-induction chemotherapy for 2nd or greater relapse of acute lymphoblastic leukaemia: a Children's Oncology Group study (ADVL1114).

The phosphatidylinositol 3-kinase (PI3K)/mammalian (or mechanistic) target of rapamycin (mTOR) signalling pathway is commonly dysregulated in acute lymphoblastic leukaemia (ALL). A phase 1 trial of the mTOR inhibitor temsirolimus in combination with UKALL R3 re-induction chemotherapy was conducted in children and adolescents with second or greater relapse of ALL. The initial temsirolimus dose level (DL1) was 10 mg/m2 weekly × 3 doses. Subsequent patient cohorts received temsirolimus 7·5 mg/m2 weekly × 3 doses (DL0) or, secondary to toxicity, 7·5 mg/m2 weekly × 2 doses (DL-1). Sixteen patients were enrolled, 15 were evaluable for toxicity. Dose-limiting toxicity (DLT) occurred at all three dose levels and included hypertriglyceridaemia, mucositis, ulceration, hypertension with reversible posterior leucoencephalopathy, elevated gamma-glutamyltransferase or alkaline phosphatase and sepsis. The addition of temsirolimus to UKALL R3 re-induction therapy resulted in excessive toxicity and was not tolerable in children with relapsed ALL. However, this regimen induced remission in seven of fifteen patients. Three patients had minimal residual disease levels <0·01%. Inhibition of PI3K signalling was detected in patients treated at all dose levels of temsirolimus, but inhibition at an early time point did not appear to correlate with clinical responses at the end of re-induction therapy.

Engagement of platelet toll-like receptor 9 by novel endogenous ligands promotes platelet hyperreactivity and thrombosis.

RATIONALE: A prothrombotic state and increased platelet reactivity are common in pathophysiological conditions associated with oxidative stress and infections. Such conditions are associated with an appearance of altered-self ligands in circulation that can be recognized by Toll-like receptors (TLRs). Platelets express a number of TLRs, including TLR9; however, the role of TLR in platelet function and thrombosis is poorly understood. OBJECTIVE: To investigate the biological activities of carboxy(alkylpyrrole) protein adducts, an altered-self ligand generated in oxidative stress, on platelet function and thrombosis. METHODS AND RESULTS: In this study we show that carboxy(alkylpyrrole) protein adducts represent novel unconventional ligands for TLR9. Furthermore, using human and murine platelets, we demonstrate that carboxy(alkylpyrrole) protein adducts promote platelet activation, granule secretion, and aggregation in vitro and thrombosis in vivo via the TLR9/MyD88 pathway. Platelet activation by TLR9 ligands induces IRAK1 and AKT phosphorylation, and it is Src kinase-dependent. Physiological platelet agonists act synergistically with TLR9 ligands by inducing TLR9 expression on the platelet surface. CONCLUSIONS: Our study demonstrates that platelet TLR9 is a functional platelet receptor that links oxidative stress, innate immunity, and thrombosis.

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-kinase and cyclic nucleotide signaling.

OBJECTIVE: Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. APPROACH AND RESULTS: Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbß3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. CONCLUSIONS: This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Impaired activation of phosphatidylinositol 3-kinase by leptin is a novel mechanism of hepatic leptin resistance in NAFLD.

BACKGROUND/AIMS: The aim of this study was to detect the levels of leptin in serum and the expression of leptin, obesity receptor (OB-R), phosphatidylinositol 3-Kinase (p85) (PI3-K p85) and phospho-Akt-kinase (Akt) in non-alcoholic fatty liver disease (NAFLD). METHODOLOGY: The expressions of leptin, OB-R and PI3-K/ Akt kinase pathway were examined by immunohistochemistry. The level of leptin in serum was measured by radioimmunoassay. RESULTS: In agreement with significantly elevated serum leptin levels in NAFLD patients (p<0.05), expression of leptin, OB-R and PI3-K (p85) was significant higher in NAFLD patients (p<0.05) compared with the control patients. In contrast, expression of Akt was significantly down-regulated in the NAFLD patients (p<0.05). Moreover, PI3-K (p85) expression was significantly, positively correlated with leptin (r= 0.365, p<0.05) but negatively correlated with Akt (r=-0.854, p<0.01). CONCLUSIONS: Leptin may be involved in NAFLD pathogenesis by activating the PI3-K/Akt kinase pathway via OB-R and the defective leptin activation of PI3-K is a novel mechanism of leptin resistance in NAFLD.

Identification of Circulating hsa_circ_0063425 and hsa_circ_0056891 as Novel Biomarkers for Detection of Type 2 Diabetes.

CONTEXT: Circular RNAs (circRNAs), which are involved in the development of diseases by regulating gene expression, have become promising novel biomarkers for diseases. OBJECTIVE: The aim of the present study was to identify the circulating circRNA biomarkers for early detection of type 2 diabetes (T2D). METHODS: The circRNA expression profiles were screened by microarray and compared between 5 new T2D cases and 5 healthy controls. The expression of candidate circRNAs that may be involved in the insulin phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway were validated by RT-qPCR in a second sample with 30 T2D cases and 30 controls. The association between circRNAs and T2D and their clinical significances were further assessed by logistic regression model, correlation analysis, and ROC curve in a large cohort comprising 313 subjects. The microRNA (miRNA) targets of circRNAs were verified by dual-luciferase reporter assay and RNA immunoprecipitation assay. RESULTS: Low expressed circ_0063425 and hsa_circ_0056891 were independent predictors of T2D, impaired fasting glucose (IFG), and insulin resistance. The 2-circRNA panel had a high diagnostic accuracy for discriminating T2D and IFG from healthy controls, especially when body mass index was integrated. miR-19a-3p and miR-1-3p were identified as the miRNA targets of hsa_circ_0063425 and hsa_circ_0056891, respectively. Significant positive correlations were found between the expression levels of AKT and hsa_circ_0063425, PI3K and hsa_circ_0056891, in the total sample and subgroups stratified by glucose levels. CONCLUSION: Downregulated hsa_circ_0063425 and hsa_circ_0056891 might contribute to the pathogenesis of T2D. They are valuable circulating biomarkers for early detection of T2D, which may be involved in regulation of PI3K/AKT signaling.

FUNDC2 regulates platelet activation through AKT/GSK-3ß/cGMP axis.

AIMS: AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. METHODS AND RESULTS: We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5'-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3ß in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3ß/cGMP axis also regulates clot retraction of platelet-rich plasma. CONCLUSION: FUNDC2 positively regulates platelet functions via AKT/GSK-3ß/cGMP signalling pathways, which provides new insight for platelet-related diseases.

Identification of Candidate Risk Factor Genes for Human Idelalisib Toxicity Using a Collaborative Cross Approach.

Idelalisib is a phosphatidylinositol 3-kinase inhibitor highly selective for the delta isoform that has shown good efficacy in treating chronic lymphocytic leukemia and follicular lymphoma. In clinical trials, however, idelalisib was associated with rare, but potentially serious liver and lung toxicities. In this study, we used the Collaborative Cross (CC) mouse population to identify genetic factors associated with the drug response that may inform risk management strategies for idelalisib in humans. Eight male mice (4 matched pairs) from 50 CC lines were treated once daily for 14 days by oral gavage with either vehicle or idelalisib at a dose selected to achieve clinically relevant peak plasma concentrations (150 mg/kg/day). The drug was well tolerated across all CC lines, and there were no observations of overt liver injury. Differences across CC lines were seen in drug concentration in plasma samples collected at the approximate Tmax on study Days 1, 7, and 14. There were also small but statistically significant treatment-induced alterations in plasma total bile acids and microRNA-122, and these may indicate early hepatocellular stress required for immune-mediated hepatotoxicity in humans. Idelalisib treatment further induced significant elevations in the total cell count of terminal bronchoalveolar lavage fluid, which may be analogous to pneumonitis observed in the clinic. Genetic mapping identified loci associated with interim plasma idelalisib concentration and the other 3 treatment-related endpoints. Thirteen priority candidate quantitative trait genes identified in CC mice may now guide interrogation of risk factors for adverse drug responses associated with idelalisib in humans.

Biomarkers Associated with Ischemic Stroke in Diabetes Mellitus Patients.

Diabetes is an established risk factor for ischemic stroke, but the associated molecular mechanisms remain to be fully elucidated. This study investigated the role of plasma and platelet microRNAs and their targeting proteins in the activation of platelets and their association with the occurrence of ischemic stroke in patients with type 2 diabetes mellitus (T2DM). Results showed that the expressions of platelet and plasma miR-144 and miR-223 were significantly altered in T2DM patients with or without ischemic stroke compared to that in healthy controls, but these changes were more significant in T2DM patients with ischemic stroke. The expressions of P2Y12 and IRS-1 as well as phosphorylation levels of IRS-1, PI3K, and Akt in platelets were significantly altered in T2DM patients with or without ischemic stroke. The expression of platelet miR-144 and miR-223 significantly correlated with their plasma levels, P2Y12 and IRS-1 expression, blood glucose concentration, and platelet activation rate. High glucose concentration significantly elevated P-selectin, miR-144 and P2Y12 expression and significantly reduced miR-223 and IRS-1 expression in UT-7 cells. Overexpression of miR-223 and blocking of miR-144 expression significantly normalized the effects of high glucose concentration in UT-7 cells. In conclusion, hyperglycemia may activate platelets through miR-144 and miR-223 to downregulate IRS-1 and upregulate P2Y12 expression in the platelets of T2DM patients through an IRS-1-PI3K-Akt signaling. Low platelet and plasma miR-223 expression in addition to high platelet and plasma miR-144 expression are risk factors for ischemic stroke in T2DM patients.

Exploration of efficacy and bleeding with combined phosphoinositide 3-kinase ß inhibition and aspirin in man.

BACKGROUND: Based on animal and human data, phosphoinositide 3-kinase (PI3K)ß is a promising antithrombotic target. However, the relation between efficacy and bleeding when combined with current antiplatelet therapies is unclear. OBJECTIVE: To strengthen the PI3Kß target validation using the short-acting inhibitor AZD6482 alone and in different combinations with P2Y12 and cyclooxygenase (COX)-1 inhibition in vitro (human platelets), in vivo (dog), and in healthy subjects. METHODS AND RESULTS: Evaluation of complete target inhibition of PI3Kß (by AZD6482), P2Y12 (by ticagrelor), and COX-1 (by aspirin) alone and in the different combinations vs. concentration responses for a panel of platelet agonists in vitro (adenosine diphosphate, collagen, thrombin receptor activating peptide) indicates that the rank order of antiplatelet efficacy is P2Y12  > PI3Kß > COX-1 as monotherapy and P2Y12 plus PI3Kß > P2Y12 plus COX-1 > PI3Kß plus COX-1 as dual therapy, with little additional effect with triple therapy. Use of a conscious dog model to assess ex vivo antiplatelet effect in parallel with bleeding time prolongation (standard incision in the ear) confirms the wide separation of efficacy vs. bleeding for PI3Kß inhibition and that this separation is reduced when combined with aspirin and more reduced when combined with clopidogrel. In healthy subjects, AZD6482, in combination with aspirin, shows a potential for greater antiplatelet potency but less bleeding potential compared with clopidogrel plus aspirin. CONCLUSIONS: PI3Kß inhibition, in comparison with P2Y12 and COX-1, delivers medium antiplatelet effect but with minimal bleeding. PI3Kß inhibition, in combination with aspirin, in healthy subjects, provides a potential for greater overall antiplatelet effect compared with clopidogrel plus aspirin, but with significantly less bleeding potential.

Abnormal expression of PI3K isoforms in patients with tobacco-related oral squamous cell carcinoma.

BACKGROUND: The phosphatidylinositol 3-kinase (PI3K) signaling regulates several cellular functions such as motility, proliferation, angiogenesis and survival. METHODS: Since there is no information on expression of PI3K isoforms in oral cancer, we studied the expression of different isoforms of PI3K (p110α, p110γ, PI3K-C2, Vps34p and p85α) in tumor samples and PBMC by RT and q-RTPCR and serum levels of PI3K p110α by SPR and ELISA techniques in 108 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 46 normal subjects. RESULTS: We observed significantly higher PI3K p110α (p<0.0001) and lower (p<0.0001) vesicular sorting protein 34p (Vps34p) mRNA both in PBMC and tissue samples of oral cancer patients as compared to the normal controls. Other PI3K isoforms did not show such change. Circulating PI3K p110α levels were higher in patients (p<0.0001) as compared to healthy subjects, the SPR data showed direct correlation with advancing stage of the disease. PI3K p110α was overexpressed in tumor samples but not in the normal buccal mucosa. CONCLUSIONS: Upregulation of circulating PI3K p110α isoform and its direct correlation with increasing tumor load in OSCC patients indicates that it may be a significant prognostic indicator and a suitable target for therapeutic/chemo-preventive strategies for tobacco-related OSCC.

P2Y12 protects platelets from apoptosis via PI3k-dependent Bak/Bax inactivation.

BACKGROUND: Platelet ADP receptor P2Y(12) is well studied and recognized as a key player in platelet activation, hemostasis and thrombosis. However, the role of P2Y(12) in platelet apoptosis remains unknown. OBJECTIVES: To evaluate the role of the P2Y(12) receptor in platelet apoptosis. METHODS: We used flow cytometry and Western blotting to assess apoptotic events in platelets treated with ABT-737 or ABT-263, and stored at 37°C, combined with P2Y(12) receptor antagonists or P2Y(12) -deficient mice. RESULTS: P2Y(12) activation attenuated apoptosis induced by ABT-737 in human and mouse platelets in vitro, evidenced by reduced phosphatidylserine (PS) exposure, diminished depolarization of mitochondrial inner transmembrane potential (ΔΨm) and decreased caspase-3 activation. Through increasing the phosphorylation level of Akt and Bad, and changing the interaction between different Bcl-2 family proteins, P2Y(12) activation inactivated Bak/Bax. This antiapoptotic effect could be abolished by P2Y(12) antagonism or PI3K inhibition. We also observed the antiapoptotic effect of P2Y(12) activation in platelets stored at 37°C. P2Y(12) activation improved the impaired activation responses of apoptotic platelets stressed by ABT-737. In platelets from mice dosed with ABT-263 in vivo, clopidogrel or deficiency of P2Y(12) receptor enhanced apoptosis along with increased Bak/Bax activation. CONCLUSIONS: This study demonstrates that P2Y(12) activation protects platelets from apoptosis via PI3k-dependent Bak/Bax inactivation, which may be physiologically important to counter the proapoptotic challenge. Our findings that P2Y(12) blockade exaggerates platelet apoptosis induced by ABT-263 (Navitoclax) also imply a novel drug interaction of ABT-263 and P2Y(12) antagonists.