Finding the "switch" in platelet activation: prediction of key mediators involved in reversal of platelet activation using a novel network biology approach.

The cAMP-protein kinase A (PKA) pathway in platelets is important for both platelet activation and inactivation. We hypothesize that proteins/processes downstream of the cAMP-PKA pathway that are regulated after platelet activation ánd subsequent inactivation can serve as a "switch" in platelet activation and inhibition. We used a STRING-based protein-protein interaction network from proteins of interest distilled from publicly available quantitative platelet proteome datasets. The protein network was integrated with biological pathway information by functional enrichment analysis, phosphorylation by PKA, and drug-target information. Functional enrichment analysis revealed biological processes related to vesicle secretion and cytoskeletal reorganization to be overrepresented among these 30 proteins coinciding with topological clusters in the network. Our method identified proteins/processes with functions related to vesicle transport, cyclin-dependent protein kinases, tight junctions, and small GTPases as potential switches in platelet activation and inhibition. Next to established enzymes in cAMP-PKA signaling, such as PDE3A, proteins with an unknown/less well-known role in platelet biology, such as Stonin-2 and ABLIM-3, emerged from our analysis as interesting candidates for reversal of platelet activation. Our method can be used to repurpose existing datasets and provide a coherent overview of mechanisms involved to predict novel connections, by visually integrating multiple datasets. SIGNIFICANCE: This article presents a novel approach of visually incorporating multiple existing tools and proteomics datasets and in doing so provides novel insight into the complex molecular mechanisms involved in platelet activation. Using our approach, we also highlight several interesting candidates for future research into pathologies with high platelet reactivity.

Transcriptomic analysis of vitamin D responses in uterine and peripheral NK cells.

Vitamin D deficiency is prevalent in pregnant women and is associated with adverse pregnancy outcomes, in particular disorders of malplacentation. The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent regulator of innate and adaptive immunity, but its immune effects during pregnancy remain poorly understood. During early gestation, the predominant immune cells in maternal decidua are uterine natural killer cells (uNK), but the responsivity of these cells to 1,25(OH)2D3 is unknown despite high levels of 1,25(OH)2D3 in decidua. Transcriptomic responses to 1,25(OH)2D3 were characterised in paired donor uNK and peripheral natural killer cells (pNK) following cytokine (CK) stimulation. RNA-seq analyses indicated 911 genes were differentially expressed in CK-stimulated uNK versus CK-stimulated pNK in the absence of 1,25(OH)2D3, with predominant differentially expressed pathways being associated with glycolysis and transforming growth factor ß (TGFß). RNA-seq also showed that the vitamin D receptor (VDR) and its heterodimer partner retinoid X receptor were differentially expressed in CK-stimulated uNK vs CK-stimulated pNK. Further analyses confirmed increased expression of VDR mRNA and protein, as well as VDR-RXR target in CK-stimulated uNK. RNA-seq analysis showed that in CK-stimulated pNK, 1,25(OH)2D3 induced 38 and suppressed 33 transcripts, whilst in CK-stimulated uNK 1,25(OH)2D3 induced 46 and suppressed 19 genes. However, multiple comparison analysis of transcriptomic data indicated that 1,25(OH)2D3 had no significant overall effect on gene expression in either CK-stimulated pNK or uNK. These data indicate that CK-stimulated uNK are transcriptionally distinct from pNK and, despite expressing abundant VDR, neither pNK nor uNK are sensitive targets for vitamin D.

Transcriptome analysis of peripheral blood mononuclear cells in human subjects following a 36 h fast provides evidence of effects on genes regulating inflammation, apoptosis and energy metabolism.

There is growing interest in the potential health benefits of diets that involve regular periods of fasting. While animal studies have provided compelling evidence that feeding patterns such as alternate-day fasting can increase longevity and reduce incidence of many chronic diseases, the evidence from human studies is much more limited and equivocal. Additionally, although several candidate processes have been proposed to contribute to the health benefits observed in animals, the precise molecular mechanisms responsible remain to be elucidated. The study described here examined the effects of an extended fast on gene transcript profiles in peripheral blood mononuclear cells from ten apparently healthy subjects, comparing transcript profiles after an overnight fast, sampled on four occasions at weekly intervals, with those observed on a single occasion after a further 24 h of fasting. Analysis of the overnight fasted data revealed marked inter-individual differences, some of which were associated with parameters such as gender and subject body mass. For example, a striking positive association between body mass index and the expression of genes regulated by type 1 interferon was observed. Relatively subtle changes were observed following the extended fast. Nonetheless, the pattern of changes was consistent with stimulation of fatty acid oxidation, alterations in cell cycling and apoptosis and decreased expression of key pro-inflammatory genes. Stimulation of fatty acid oxidation is an expected response, most likely in all tissues, to fasting. The other processes highlighted provide indications of potential mechanisms that could contribute to the putative beneficial effects of intermittent fasting in humans.

Cardiac contractile dysfunction in insulin-resistant rats fed a high-fat diet is associated with elevated CD36-mediated fatty acid uptake and esterification.

AIMS/HYPOTHESIS: Changes in cardiac substrate utilisation leading to altered energy metabolism may underlie the development of diabetic cardiomyopathy. We studied cardiomyocyte substrate uptake and utilisation and the role of the fatty acid translocase CD36 in relation to in vivo cardiac function in rats fed a high-fat diet (HFD). METHODS: Rats were exposed to an HFD or a low-fat diet (LFD). In vivo cardiac function was monitored by echocardiography. Substrate uptake and utilisation were determined in isolated cardiomyocytes. RESULTS: Feeding an HFD for 8 weeks induced left ventricular dilation in the systolic phase and decreased fractional shortening and the ejection fraction. Insulin-stimulated glucose uptake and proline-rich Akt substrate 40 phosphorylation were 41% (p < 0.001) and 45% (p < 0.05) lower, respectively, in cardiomyocytes from rats on the HFD. However, long-chain fatty acid (LCFA) uptake was 1.4-fold increased (p < 0.001) and LCFA esterification into triacylglycerols and phospholipids was increased 1.4- and 1.5-fold, respectively (both p < 0.05), in cardiomyocytes from HFD compared with LFD hearts. In the presence of the CD36 inhibitor sulfo-N-succinimidyloleate, LCFA uptake and esterification were similar in LFD and HFD cardiomyocytes. In HFD hearts CD36 was relocated to the sarcolemma, and basal phosphorylation of a mediator of CD36-trafficking, i.e. protein kinase B (PKB/Akt), was increased. CONCLUSIONS/INTERPRETATION: Feeding rats an HFD induced cardiac contractile dysfunction, which was accompanied by the relocation of CD36 to the sarcolemma, and elevated basal levels of phosphorylated PKB/Akt. The permanent presence of CD36 at the sarcolemma resulted in enhanced rates of LCFA uptake and myocardial triacylglycerol accumulation, and may contribute to the development of insulin resistance and diabetic cardiomyopathy.

Divergent effects of rosiglitazone on protein-mediated fatty acid uptake in adipose and in muscle tissues of Zucker rats.

Thiazolidinediones (TZDs) increase tissue insulin sensitivity in diabetes. Here, we hypothesize that, in adipose tissue, skeletal muscle, and heart, alterations in protein-mediated FA uptake are involved in the effect of TZDs. As a model, we used obese Zucker rats, orally treated for 16 days with 5 mg rosiglitazone (Rgz)/kg body mass/day. In adipose tissue from Rgz-treated rats, FA uptake capacity increased by 2.0-fold, coinciding with increased total contents of fatty acid translocase (FAT/CD36; 2.3-fold) and fatty acid transport protein 1 (1.7-fold) but not of plasmalemmal fatty acid binding protein, whereas only the plasmalemmal content of FAT/CD36 was changed (increase of 1.7-fold). The increase in FA uptake capacity of adipose tissue was associated with a decline in plasma FA and triacylglycerols (TAGs), suggesting that Rgz treatment enhanced plasma FA extraction by adipocytes. In obese hearts, Rgz treatment had no effect on the FA transport system, yet the total TAG content decreased, suggesting enhanced insulin sensitivity. Also, in skeletal muscle, the FA transport system was not changed. However, the TAG content remained unaltered in skeletal muscle, which coincided with increased cytoplasmic adipose-type FABP content, suggesting that increased extramyocellular TAGs mask the decline of intracellular TAG in muscle. In conclusion, our study implicates FAT/CD36 in the mechanism by which Rgz increases tissue insulin sensitivity.

Increased FAT (fatty acid translocase)/CD36-mediated long-chain fatty acid uptake in cardiac myocytes from obese Zucker rats.

Disturbed cardiac lipid homoeostasis in obesity is regarded as a key player in the development of cardiovascular diseases. In this study, we show that FAT (fatty acid translocase)/CD36-mediated LCFA (long-chain fatty acid) uptake in cardiac myocytes from young adult obese Zucker rats is markedly increased, but insensitive to insulin. Basal and insulin-induced glucose uptake rates in these myocytes are not changed, suggesting that during the development from obesity to hyperglycaemic Type II diabetes, alterations in cardiac LCFA uptake precede alterations in cardiac glucose uptake.

Effects of cAMP modulators on long-chain fatty-acid uptake and utilization by electrically stimulated rat cardiac myocytes.

Recently, we established that cellular contractions increase long-chain fatty-acid (FA) uptake by cardiac myocytes. This increase is dependent on the transport function of an 88 kDa membrane FA transporter, FA translocase (FAT/CD36), and, in analogy to skeletal muscle, is likely to involve its translocation from an intracellular pool to the sarcolemma. In the present study, we investigated whether cAMP-dependent signalling is involved in this translocation process. Isoproterenol, dibutyryl-cAMP and the phosphodiesterase (PDE) inhibitor, amrinone, which markedly raised the intracellular cAMP level, did not affect cellular FA uptake, but influenced the fate of intracellular FAs by directing these to mitochondrial oxidation in electrostimulated cardiac myocytes. The PDE inhibitors 3-isobutyl-1-methylxanthine, milrinone and dipyridamole each significantly stimulated FA uptake as well as intracellular cAMP levels, but these effects were quantitatively unrelated. The stimulatory effects of these PDE inhibitors were antagonized by sulpho- N -succinimidylpalmitate, indicating the involvement of FAT/CD36, albeit that the different PDE inhibitors use different molecular mechanisms to stimulate FAT/CD36-mediated FA uptake. Notably, 3-isobutyl-1-methylxanthine and milrinone increased the intrinsic activity of FAT/CD36, possibly through its covalent modification, and dipyridamole induces translocation of FAT/CD36 to the sarcolemma. Elevation of intracellular cGMP, but not of cAMP, by the PDE inhibitor zaprinast did not have any effect on FA uptake and metabolism by cardiac myocytes. The stimulatory effects of PDE inhibitors on cardiac FA uptake should be considered when applying these agents in clinical medicine.