Bilberry Supplementation after Myocardial Infarction Decreases Microvesicles in Blood and Affects Endothelial Vesiculation

Scope: Diet rich in bilberries is considered cardioprotective, but the mechanisms of action are poorly understood. Cardiovascular disease is characterized by increased proatherogenic status and high levels of circulating microvesicles (MVs). In an open-label study patients with myocardial infarction receive an 8 week dietary supplementation with bilberry extract (BE). The effect of BE on patient MV levels and its influence on endothelial vesiculation in vitro is investigated. Methods and results: MVs are captured with acoustic trapping and platelet-derived MVs (PMVs), as well as endothelial-derived MVs (EMVs) are quantified with flow cytometry. The in vitro effect of BE on endothelial extracellular vesicle (EV) release is examined using endothelial cells and calcein staining. The mechanisms of BE influence on vesiculation pathways are studied by Western blot and qRT-PCR. Supplementation with BE decreased both PMVs and EMVs. Furthermore, BE reduced endothelial EV release, Akt phosphorylation, and vesiculation-related gene transcription. It also protects the cells from P2X7-induced EV release and increase in vesiculation-related gene expression. Conclusion: BE supplementation improves the MV profile in patient blood and reduces endothelial vesiculation through several molecular mechanisms related to the P2X7 receptor. The findings provide new insight into the cardioprotective effects of bilberries

A MATTER OF NUTRIENT EXCESS AND SHEAR STRESS

The metabolic syndrome is a clustering of several risk factors associated with diabetes and cardiovascular disease. Diabetes is a global problem with 90% of patients suffering from type 2 diabetes (T2D), which is attributable to various factors including genetic predispositions and lifestyle. In T2D, there is a reduction in β cell mass and function, and impaired glucose homeostasis. It is often associated with insulin resistance in target tissues and has a profound influence on vascular health. This, thesis investigates the novel role of cocaine and amphetamine regulated transcript (CART) in the pancreatic β cells and purinergic signaling mechanisms in the vascular endothelial cells. Using rat pancreatic clonal β cell lines (INS-1 832/13) and rat islets, we provide evidence for a novel role of CART in β cell survival and proliferation. Exogenous addition of CART 55-102 attenuated glucotoxicity-induced β cell death. This effect of CART was likely due to the activation of signaling molecules important for cell survival, namely, CREB, IRS-2, PKB, FoxO1, p44/42-MAPK, and p90RSK. Furthermore, the pharmacological inhibition of the kinases blocked the proliferative effect of CART on β cells. In conclusion, CART 55-102 protects β cells against glucotoxicity and promotes proliferation and may thereby contribute to the preservation of β cell mass. Physical inactivity and metabolic risk factors together form a continuum that over time negatively affects endothelial function. The vascular endothelium is exposed to fluid shear stress from flowing blood and maintains vascular homeostasis. The resident endothelial cells express a repertoire of specialized purinergic receptors that respond to extracellular nucleotides mediating various physiological responses. In human vascular endothelial cells, both ATPγS and shear stress increased KLF2, a transcription factor important for atheroprotection, in part via the P2X4 receptor. Additionally, shear stress-induced endothelial cell alignment and cytoskeletal remodeling, as seen in atheroprotective regions of the vascular tree, was found to be P2Y2 receptor-dependent. Using pharmacological inhibitors of P2X7 and P2X4, we observed differential effects of these receptors in inhibiting inflammatory genes, reactive oxygen species, and leukocyte adhesion in endothelial cells exposed to high glucose and palmitate. In addition, we demonstrate a novel role of UTP and ATP in regulating miR-22 transcription that inhibits ICAM-1 and leukocyte-endothelial adhesion, an effect possibly mediated by P2Y2 receptors. In conclusion, this thesis provides a molecular basis for treatment strategies, albeit at a cellular level, aiming at (i) preserving β cell mass and function and (ii) enhancing vascular health that range from lifestyle interventions to specific pharmacological therapies

The ATP Receptors P2X7 and P2X4 Modulate High Glucose and Palmitate-Induced Inflammatory Responses in Endothelial Cells.

Endothelial cells lining the blood vessels are principal players in vascular inflammatory responses. Dysregulation of endothelial cell function caused by hyperglycemia, dyslipidemia, and hyperinsulinemia often result in impaired vasoregulation, oxidative stress, inflammation, and altered barrier function. Various stressors including high glucose stimulate the release of nucleotides thus initiating signaling via purinergic receptors. However, purinergic modulation of inflammatory responses in endothelial cells caused by high glucose and palmitate remains unclear. In the present study, we investigated whether the effect of high glucose and palmitate is mediated by P2X7 and P2X4 and if they play a role in endothelial cell dysfunction. Transcript and protein levels of inflammatory genes as well as reactive oxygen species production, endothelial-leukocyte adhesion, and cell permeability were investigated in human umbilical vein endothelial cells exposed to high glucose and palmitate. We report high glucose and palmitate to increase levels of extracellular ATP, expression of P2X7 and P2X4, and inflammatory markers. Both P2X7 and P2X4 antagonists inhibited high glucose and palmitate-induced interleukin-6 levels with the former having a significant effect on interleukin-8 and cyclooxygenase-2. The effect of the antagonists was confirmed with siRNA knockdown of the receptors. In addition, P2X7 mediated both high glucose and palmitate-induced increase in reactive oxygen species levels and decrease in endothelial nitric oxide synthase. Blocking P2X7 inhibited high glucose and palmitate-induced expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 as well as leukocyte-endothelial cell adhesion. Interestingly, high glucose and palmitate enhanced endothelial cell permeability that was dependent on both P2X7 and P2X4. Furthermore, antagonizing the P2X7 inhibited high glucose and palmitate-mediated activation of p38-mitogen activated protein kinase. These findings support a novel role for P2X7 and P2X4 coupled to induction of inflammatory molecules in modulating high glucose and palmitate-induced endothelial cell activation and dysfunction

Distribution of melatonin receptors in murine pancreatic islets.

Melatonin has multiple receptor-dependent and receptor-independent functions. At the cell membrane, melatonin interacts with its receptors MT1 and MT2, which are expressed in numerous tissues. Genome-wide association studies have recently shown that the MTNR1B/MT2 receptor may be involved in the pathogenesis of type 2 diabetes mellitus. In line with these findings, expression of melatonin receptors has been shown in mouse, rat, and human pancreatic islets. MT1 and MT2 are G-protein-coupled receptors and are proposed to exert inhibitory effects on insulin secretion. Here, we show by immunocytochemistry that these membrane melatonin receptors have distinct locations in the mouse islet. MT1 is expressed in α-cells while MT2 is located to the β-cells. These findings help to unravel the complex machinery underlying melatonin's role in the regulation of islet function

Cocaine- and Amphetamine-regulated Transcript (CART) Protects Beta Cells against Glucotoxicity and Increases Cell Proliferation

Cocaine and amphetamine-regulated transcript (CART) is an islet peptide that promotes glucose stimulated insulin secretion in beta cells via cAMP/PKA-dependent pathways. In addition, CART is a regulator of neuronal survival. In this study, we examined the effect of exogenous CART 55-102 on beta cell viability and dissected its signaling mechanisms. Evaluation of DNA fragmentation and chromatin condensation revealed that CART 55-102 reduced glucotoxicity-induced apoptosis in both INS-1 (832/13) cells and isolated rat islets. Furthermore, glucotoxicity in INS-1 (832/13) cells caused a 50% reduction of endogenous CART protein. In addition, CART increased proliferation in INS-1 (832/13) cells; an effect that was blocked by PKA, PKB, and MEK1 inhibitors. Further, in INS-1 (832/13) cells and isolated rat islets, CART induced phosphorylation of CREB, IRS-2, PKB, FoxO1, p44/42 MAPK, and p90RSK; all key mediators of cell survival and proliferation. Thus, we demonstrate that CART 55-102 protects beta cells against glucotoxicity and promotes proliferation. Taken together our data points to the potential use of CART in therapeutic interventions targeted at enhancing functional beta cell mass and long-term insulin secretion in T2D

P2X7 and P2X4 antagonists block high glucose and palmitate-induced expression of inflammatory genes.

<p>qRT-PCR analysis shows high glucose and palmitate-induced (24 h) transcript levels of <i>CASP1</i> (A), <i>IL-1β</i> (B), <i>IL-6</i> (C), <i>PTGS2</i> (D), and <i>IL-8</i> (E) in the presence or absence of the P2X7 (AZ11645373) and P2X4 (PSB-12253) antagonists. The transcript levels were normalized to the housekeeping gene, <i>PPIA</i>. n = 3 to 4 independent experiments each done in replicates; *<i>p</i> ≤ 0.05.</p