Loss of the insulin receptor in murine megakaryocytes/platelets causes thrombocytosis and alterations in IGF signalling.

AIMS: Patients with conditions that are associated with insulin resistance such as obesity, type 2 diabetes mellitus, and polycystic ovary syndrome have an increased risk of thrombosis and a concurrent hyperactive platelet phenotype. Our aim was to determine whether insulin resistance of megakaryocytes/platelets promotes platelet hyperactivation. METHODS AND RESULTS: We generated a conditional mouse model where the insulin receptor (IR) was specifically knocked out in megakaryocytes/platelets and performed ex vivo platelet activation studies in wild-type (WT) and IR-deficient platelets by measuring aggregation, integrin αIIbβ3 activation, and dense and α-granule secretion. Deletion of IR resulted in an increase in platelet count and volume, and blocked the action of insulin on platelet signalling and function. Platelet aggregation, granule secretion, and integrin αIIbβ3 activation in response to the glycoprotein VI (GPVI) agonist collagen-related peptide (CRP) were significantly reduced in platelets lacking IR. This was accompanied by a reduction in the phosphorylation of effectors downstream of GPVI. Interestingly, loss of IR also resulted in a reduction in insulin-like growth factor-1 (IGF-1)- and insulin-like growth factor-2 (IGF-2)-mediated phosphorylation of IRS-1, Akt, and GSK3β and priming of CRP-mediated platelet activation. Pharmacological inhibition of IR and the IGF-1 receptor in WT platelets recapitulated the platelet phenotype of IR-deficient platelets. CONCLUSIONS: Deletion of IR (i) increases platelet count and volume, (ii) does not cause platelet hyperactivity, and (iii) reduces GPVI-mediated platelet function and platelet priming by IGF-1 and IGF-2

Census 2010 Demographic Profile: Jefferson County

This demographic profile describes characteristics of the local and state population based on results from the 2010 Census. The decennial census is an official enumeration, or count, of all residents on April 1st of the census year. The results of the census provide us with information about basic demographic characteristics of the population, including age, race, ethnicity, household composition, housing occupancy, and housing tenure

Critical roles for the phosphatidylinositide 3-kinase isoforms p110β and p110γ in thrombopoietin-mediated priming of platelet function

Abstract Thrombopoietin (TPO) enhances platelet activation through activation of the tyrosine kinase; JAK2 and the lipid kinase phosphatidylinositide 3-kinase (PI3K). The aim of our study was to identify the PI3K isoforms involved in mediating the effect of TPO on platelet function and elucidate the underlying mechanism. We found that p110β plays an essential role in TPO-mediated (i) priming of protease-activated receptor (PAR)-mediated integrin αIIbβ3 activation and α-granule secretion, (ii) synergistic enhancement of PAR-mediated activation of the small GTPase RAP1, a regulator of integrin activation and (iii) phosphorylation of the PI3K effector Akt. More importantly, the synergistic effect of TPO on phosphorylation of extracellular-regulated kinase (ERK1/2) and thromboxane (TxA2) synthesis was dependent on both p110β and p110γ. p110β inhibition/deletion, or inhibition of p110γ, resulted in a partial reduction, whereas inhibiting both p110β and p110γ completely prevented the synergistic effect of TPO on ERK1/2 phosphorylation and TxA2 synthesis. The latter was ablated by inhibition of MEK, but not p38, confirming a role for ERK1/2 in regulating TPO-mediated increases in TxA2 synthesis. In conclusion, the synergistic effect of TPO on RAP1 and integrin activation is largely mediated by p110β, whereas p110β and p110γ contribute to the effect of TPO on ERK1/2 phosphorylation and TxA2 formation

Liver transplantation for type I and type IV glycogen storage disease

Progressive liver failure or hepatic complications of the primary disease led to orthotopic liver transplantation in eight children with glycogen storage disease over a 9-year period. One patient had glycogen storage disease (GSD) type I (von Gierke disease) and seven patients had type IV GSD (Andersen disease). As previously reported [19], a 16.5-year-old-girl with GSD type I was successfully treated in 1982 by orthotopic liver transplantation under cyclosporine and steroid immunosuppression. The metabolic consequences of the disease have been eliminated, the renal function and size have remained normal, and the patient has lived a normal young adult life. A late portal venous thrombosis was treated successfully with a distal splenorenal shunt. Orthotopic liver transplantation was performed in seven children with type N GSD who had progressive hepatic failure. Two patients died early from technical complications. The other five have no evidence of recurrent hepatic amylopectinosis after 1.1–5.8 postoperative years. They have had good physical and intellectual maturation. Amylopectin was found in many extrahepatic tissues prior to surgery, but cardiopathy and skeletal myopathy have not developed after transplantation. Postoperative heart biopsies from patients showed either minimal amylopectin deposits as long as 4.5 years following transplantation or a dramatic reduction in sequential biopsies from one patient who initially had dense myocardial deposits. Serious hepatic derangement is seen most commonly in types T and IV GSD. Liver transplantation cures the hepatic manifestations of both types. The extrahepatic deposition of abnormal glycogen appears not to be problematic in type I disease, and while potentially more threatening in type IV disease, may actually exhibit signs of regression after hepatic allografting

Expression of Regulatory Platelet MicroRNAs in Patients with Sickle Cell Disease

Background: Increased platelet activation in sickle cell disease (SCD) contributes to a state of hypercoagulability and confers a risk of thromboembolic complications. The role for post-transcriptional regulation of the platelet transcriptome by microRNAs (miRNAs) in SCD has not been previously explored. This is the first study to determine whether platelets from SCD exhibit an altered miRNA expression profile. Methods and Findings: We analyzed the expression of miRNAs isolated from platelets from a primary cohort (SCD = 19, controls = 10) and a validation cohort (SCD = 7, controls = 7) by hybridizing to the Agilent miRNA microarrays. A dramatic difference in miRNA expression profiles between patients and controls was noted in both cohorts separately. A total of 40 differentially expressed platelet miRNAs were identified as common in both cohorts (p-value 0.05, fold change>2) with 24 miRNAs downregulated. Interestingly, 14 of the 24 downregulated miRNAs were members of three families - miR-329, miR-376 and miR-154 - which localized to the epigenetically regulated, maternally imprinted chromosome 14q32 region. We validated the downregulated miRNAs, miR-376a and miR-409-3p, and an upregulated miR-1225-3p using qRT-PCR. Over-expression of the miR-1225-3p in the Meg01 cells was followed by mRNA expression profiling to identify mRNA targets. This resulted in significant transcriptional repression of 1605 transcripts. A combinatorial approach using Meg01 mRNA expression profiles following miR-1225-3p overexpression, a computational prediction analysis of miRNA target sequences and a previously published set of differentially expressed platelet transcripts from SCD patients, identified three novel platelet mRNA targets: PBXIP1, PLAGL2 and PHF20L1. Conclusions: We have identified significant differences in functionally active platelet miRNAs in patients with SCD as compared to controls. These data provide an important inventory of differentially expressed miRNAs in SCD patients and an experimental framework for future studies of miRNAs as regulators of biological pathways in platelets. © 2013 Jain et al

Global identification of genes and pathways regulated by Akt during activation of T helper cells

We previously demonstrated that Akt differentially modulated a subset of NF-kB target genes during T cell activation. In the current study, we further explored the broader effects of Akt inhibition on T cell gene induction. Global microarray analysis was used to characterize T helper cell transcriptional responses following antigen receptor stimulation in the absence or presence of Akti1/2 (an allosteric inhibitor which targets Akt1 and Akt2), to identify novel targets dependent upon Akt and obtain a more comprehensive view of Akt-sensitive genes in Th2 helper T cells. Pathway analysis of microarray data from a CD4+ Th2 T cell line revealed effects on gene networks involving ribosomal and T cell receptor signaling pathways associated with Akti1/2 treatment. Using real-time PCR analysis, we validated the differential regulation of several genes in these pathways, including Ier3, Il13, Egr1, Ccl1 and Ccl4, among others. Additionally, transcription factor target gene (TFactS) analysis revealed that NF-kB and Myc were the most significantly enriched transcription factors among Akt-dependent genes after T cell receptor and CD28 stimulation. Akt activation elicited increases in the enrichment of NF-kB- and Myc-targeted genes. The present study has identified a diverse set of genes, and possible mechanisms for their regulation, that are dependent on Akt during T cell activation

Perifosine as a Potential Novel Anti-Cancer Agent Inhibits EGFR/MET-AKT Axis in Malignant Pleural Mesothelioma

PI3K/AKT signalling pathway is aberrantly active and plays a critical role for cell cycle progression of human malignant pleural mesothelioma (MMe) cells. AKT is one of the important cellular targets of perifosine, a novel bio-available alkylphospholipid that has displayed significant anti-proliferative activity in vitro and in vivo in several human tumour model systems and is currently being tested in clinical trials.We tested Perifosine activity on human mesothelial cells and different mesothelioma cell lines, in order to provide evidence of its efficacy as single agent and combined therapy.We demonstrate here that perifosine, currently being evaluated as an anti-cancer agent in phase 1 and 2 clinical trials, caused a dose-dependent reduction of AKT activation, at concentrations causing MMe cell growth arrest. In this study we firstly describe that MMe cells express aside from AKT1 also AKT3 and that either the myristoylated, constitutively active, forms of the two proteins, abrogated perifosine-mediated cell growth inhibition. Moreover, we describe here a novel mechanism of perifosine that interferes, upstream of AKT, affecting EGFR and MET phosphorylation. Finally, we demonstrate a significant increase in cell toxicity when MMe cells were treated with perifosine in combination with cisplatin.This study provides a novel mechanism of action of perifosine, directly inhibiting EGFR/MET-AKT1/3 axis, providing a rationale for a novel translational approach to the treatment of MMe