The human cytomegalovirus-encoded G protein- coupled receptor UL33 exhibits oncomodulatory properties

Herpesviruses can rewire cellular signaling in host cells by expressing viral G protein- coupled receptors (GPCRs). These viral receptors exhibit homology to human chemokine receptors, but some display constitutive activity and promiscuous G protein coupling. Human cytomegalovirus (HCMV) has been detected in multiple cancers, including glioblastoma, and its genome encodes four GPCRs. One of these receptors, US28, is expressed in glioblastoma and possesses constitutive activity and oncomodulatory properties. UL33, another HCMV-encoded GPCR, also displays constitutive signaling via Gαq, Gαi, and Gαs proteins. However, little is known about the nature and functional effects of UL33-driven signaling. Here, we assessed UL33's signaling repertoire and oncomodulatory potential. UL33 activated multiple proliferative, angiogenic, and inflammatory signaling pathways in HEK293T and U251 glioblastoma cells. Notably, upon infection, UL33 contributed to HCMV-mediated STAT3 activation. Moreover, UL33 increased spheroid growth in vitro and accelerated tumor growth in different in vivo tumor models, including an orthotopic glioblastoma xenograft model. UL33-mediated signaling was similar to that stimulated by US28; however, UL33-induced tumor growth was delayed. Additionally, the spatiotemporal expression of the two receptors only partially overlapped in HCMV-infected glioblastoma cells. In conclusion, our results unveil that UL33 has broad signaling capacity and provide mechanistic insight into its functional effects. UL33, like US28, exhibits oncomodulatory properties, elicited via constitutive activation of multiple signaling pathways. UL33 and US28 might contribute to HCMV's oncomodulatory effects through complementing and converging cellular signaling, and hence UL33 may represent a promising drug target in HCMV-associated malignancies

Homocysteine-induced cardiomyocyte apoptosis and plasma membrane flip-flop are independent of S-adenosylhomocysteine: a crucial role for nuclear p47(phox).

Item does not contain fulltextWe previously found that homocysteine (Hcy) induced plasma membrane flip-flop, apoptosis, and necrosis in cardiomyocytes. Inactivation of flippase by Hcy induced membrane flip-flop, while apoptosis was induced via a NOX2-dependent mechanism. It has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in hyperhomocysteinemia (HHC)-induced pathogenesis of cardiovascular disease. Therefore, we evaluated whether the observed cytotoxic effect of Hcy in cardiomyocytes is SAH dependent. Rat cardiomyoblasts (H9c2 cells) were treated under different conditions: (1) non-treated control (1.5 nM intracellular SAH with 2.8 muM extracellular L -Hcy), (2) incubation with 50 muM adenosine-2,3-dialdehyde (ADA resulting in 83.5 nM intracellular SAH, and 1.6 muM extracellular L -Hcy), (3) incubation with 2.5 mM D, L -Hcy (resulting in 68 nM intracellular SAH and 1513 muM extracellular L -Hcy) with or without 10 muM reactive oxygen species (ROS)-inhibitor apocynin, and (4) incubation with 100 nM, 10 muM, and 100 muM SAH. We then determined the effect on annexin V/propodium iodide positivity, flippase activity, caspase-3 activity, intracellular NOX2 and p47(phox) expression and localization, and nuclear ROS production. In contrast to Hcy, ADA did not induce apoptosis, necrosis, or membrane flip-flop. Remarkably, both ADA and Hcy induced a significant increase in nuclear NOX2 expression. However, in contrast to ADA, Hcy additionally induced nuclear p47(phox) expression, increased nuclear ROS production, and inactivated flippase. Incubation with SAH did not have an effect on cell viability, nor on flippase activity, nor on nuclear NOX2-, p47phox expression or nuclear ROS production. HHC-induced membrane flip-flop and apoptosis in cardiomyocytes is due to increased Hcy levels and not primarily related to increased intracellular SAH, which plays a crucial role in nuclear p47(phox) translocation and subsequent ROS production.1 december 201

Therapeutic Use of Microbubbles and Ultrasound in Acute Peripheral Arterial Thrombosis: A Systematic Review

Catheter-directed thrombolysis (CDT) for acute peripheral arterial occlusion is time consuming and carries a risk of major hemorrhage. Contrast-enhanced sonothrombolysis (CEST) might enhance outcomes compared with standard CDT. In the study described here, we systematically reviewed all in vivo studies on contrast-enhanced sonothrombolysis in a setting of arterial thrombosis. A systematic search of the PubMed, Embase, Cochrane Library and Web of Science databases was conducted. Two reviewers independently performed the study selection, quality assessment and data extraction. Primary outcomes were recanalization rate and thrombus weight. Secondary outcome was any possible adverse event. The 35 studies included in this review were conducted in four different (pre)clinical settings: ischemic stroke, myocardial infarction, (peripheral) arterial thrombosis and arteriovenous graft occlusion. Because of the high heterogeneity among the studies, it was not possible to conduct a meta-analysis. In almost all studies, recanalization rates were higher in the group that underwent a form of CEST. One study was terminated early because of a higher incidence of intracranial hemorrhage. Studies on CEST suggest that adding microbubbles and ultrasound to standard intra-arterial CDT is safe and might improve outcomes in acute peripheral arterial thrombosis. Further research is needed before CEST can be implemented in daily practice

Reactive Oxygen Species Precede Protein Kinase C-δ Activation Independent of Adenosine Triphosphate-sensitive Mitochondrial Channel Opening in Sevoflurane-induced Cardioprotection

Background: In the current study, the authors investigated the distinct role and relative order of protein kinase C (PKC)-δ adenosine triphosphate-sensitive mitochondrial K + (mito K + ATP) channels, and reactive oxygen species (ROS) in the signal transduction of sevoflurane-induced cardioprotection and specifically addressed their mechanistic link. Methods: Isolated rat trabeculae were preconditioned with 3.8% sevoflurane and subsequently subjected to an ischemic protocol by superfusion of trabeculae with hypoxic, glucose-free buffer (40 min) followed by 60 min of reperfusion. In addition, the acute affect of sevoflurane on PKC-δ and PKC-δ translocation and nitrotyrosine formation was established with use of immunofluorescent analysis. The inhibitors chelerythrine (6 μM), rottlerin (1 μM), 5-hydroxydecanoic acid sodium (100 μM), and n-(2-mercaptopropionyl)-glycine (300 μM) were used to study the particular role of PKC, PKC-δ, mito K + ATP, and ROS in sevoflurane-related intracellular signaling. Results: Preconditioning of trabeculae with sevoflurane preserved contractile function after ischemia. This contractile preservation was dependent on PKC-δ activation, mito K + ATP channel opening, and ROS production. In addition, on acute stimulation by sevoflurane, PKC-δ but not PKC-ε translocated to the sarcolemmal membrane. This translocation was inhibited by PKC inhibitors and ROS scavenging but not by inhibition of mito K + ATP channels. Furthermore, sevoflurane directly induced nitrosylation of sarcolemmal proteins, suggesting the formation of peroxynitrite. Conclusions: In sevoflurane-induced cardioprotection, ROS release but not mito K + ATP channel opening precedes PKC-δ activation. Sevoflurane induces sarcolemmal nitrotyrosine formation, which might be involved in the recruitment of PKC-δ to the cell membrane

Astrocytic Abeta1-42 uptake is determined by Abeta-aggregation state and the presence of amyloid-associated proteins

Intracerebral accumulation of amyloid-beta (Abeta) leading to Abeta plaque formation, is the main hallmark of Alzheimer's disease and might be caused by defective Abeta-clearance. We previously found primary human astrocytes and microglia able to bind and ingest Abeta1-42 in vitro, which appeared to be limited by Abeta1-42 fibril formation. We now confirm that astrocytic Abeta-uptake depends on size and/or composition of Abeta-aggregates as astrocytes preferably take up oligomeric Abeta over fibrillar Abeta. Upon exposure to either fluorescence-labelled Abeta1-42 oligomers (Abeta(oligo)) or fibrils (Abeta(fib)), a larger (3.7 times more) proportion of astrocytes ingested oligomers compared to fibrils, as determined by flow cytometry. Abeta-internalization was verified using confocal microscopy and live-cell imaging. Neither uptake of Abeta(oligo) nor Abeta(fib), triggered proinflammatory activation of the astrocytes, as judged by quantification of interleukin-6 and monocyte-chemoattractant protein-1 release. Amyloid-associated proteins, including alpha1-antichymotrypsin (ACT), serum amyloid P component (SAP), C1q and apolipoproteins E (ApoE) and J (ApoJ) were earlier found to influence Abeta-aggregation. Here, astrocytic uptake of Abeta(fib) increased when added to the cells in combination with SAP and C1q (SAP/C1q), but was unchanged in the presence of ApoE, ApoJ and ACT. Interestingly, ApoJ and ApoE dramatically reduced the number of Abeta(oligo)-positive astrocytes, whereas SAP/C1q slightly reduced Abeta(oligo) uptake. Thus, amyloid-associated proteins, especially ApoJ and ApoE, can alter Abeta-uptake in vitro and hence may influence Abeta clearance and plaque formation in vivo. (c) 2010 Wiley-Liss, In

Increased local delivery of antagomir therapeutics to the rodent myocardium using ultrasound and microbubbles

Recent developments in microRNA (miRNA) research have identified these as important mediators in the pathophysiological response upon myocardial infarction (MI). Specific miRNAs can inhibit the translation of entire groups of mRNAs, which are involved in specific processes in the pathophysiology after MI, e.g. the fibrotic, apoptotic or angiogenic response. By modulating miRNAs in the heart, these processes can be tuned to improve cardiac function. Antagomirs are effective miRNA-inhibitors, but have a low myocardial specificity and cardiac antagomir treatment therefore requires high doses, which causes side effects. In the present study, ultrasound-triggered microbubble destruction (UTMD) was studied to increase specific delivery of antagomir to the myocardium. Healthy control mice were treated with UTMD and sacrificed at 30min, 24h and 48h, after which antagomir delivery in the heart was analyzed, both qualitatively and quantitatively. Additionally, potential harmful effects of treatment were analyzed by monitoring ECG, analyzing neutrophil invasion and cell death in the heart, and measuring troponin I after treatment. Finally, UTMD was tested for delivery of antagomir in a model of ischemia-reperfusion (I/R) injury. We found that UTMD can significantly increase local antagomir delivery to the non-ischemic heart with modest side-effects like neutrophil invasion without causing apoptosis. Delivered antagomirs enter cardiomyocytes within 30min after treatment and remains there for at least 48h. Interestingly, after I/R injury antagomir already readily enters the infarcted zone and we observed no additional benefit of UTMD for antagomir delivery. This study is the first to explore cardiac antagomir delivery using UTMD. In addition, it is the first to study tissue distribution of short RNA based therapeutics (~22 base pairs) at both the cellular and organ levels after UTMD to the heart in general. In summary, UTMD provides a myocardial delivery strategy for non-vascular permeable cardiac conditions later in the I/R response or chronic conditions like cardiac hypertrophy

Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild-type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate hos

The Potential Role of Neutrophil Gelatinase-Associated Lipocalin in the Development of Abdominal Aortic Aneurysms

Background: In abdominal aortic aneurysm (AAA), pathophysiology deterioration of the medial aortic layer plays a critical role. Key players in vessel wall degeneration are reactive oxygen species (ROS), smooth muscle cell apoptosis, and extracellular matrix degeneration by matrix metalloproteinase-9 (MMP-9). Lipocalin-2, also neutrophil gelatinase-associated lipocalin (NGAL), is suggested to be involved in these degenerative processes in other cardiovascular diseases. We aimed to further investigate the role of NGAL in AAA development and rupture. Methods: In this observational study, aneurysm tissue and blood of ruptured (n = 13) AAA patients were investigated versus nonruptured (n = 26) patients. Nondilated aortas (n = 5) from deceased patients and venous blood from healthy volunteers (n = 10) served as controls. NGAL concentrations in tissue and blood were measured by enzyme-linked immunosorbent assay and immunofluorescence microscopy. Nitrotyrosine (marker of ROS), MMP-9, and caspase-3 (marker of apoptosis) in aneurysm tissue were measured by immunofluorescence microscopy. AAA expansion rates were calculated retrospectively. Results: NGAL (in μg/mL) blood concentration in ruptured AAA was 46 (range 22–122) vs. 26 (range 6–55) in nonruptured AAA (P < 0.01) and 14 (range 12–22) in controls (P < 0.01). In the aneurysm wall of ruptured AAA, NGAL concentration was 4.7 (range 1.4–25) vs. 4.4 (range 0.2–14) in nonruptured AAA (not significant) and 1.8 (range 1.2–2.7) in nondilated aortas (P = 0.04). In the medial layer, NGAL correlated positively with nitrotyrosine (Rs = 0.80, P < 0.01), MMP-9 (Rs = 0.56, P = 0.02), and caspase-3 (Rs = 0.75, P = 0.01). NGAL did not correlate to AAA expansion rate in blood or tissue (P = 0.34 and P = 0.95, respectively). Conclusions: This study demonstrates that NGAL blood concentration is higher in ruptured AAA patients than in nonruptured AAA. NGAL expression in the AAA wall is also higher than in nondilated aorta. Furthermore, its expression is associated with factors of vessel wall deterioration. Based on our study results, we could not determine NGAL as a biomarker for AAA growth or rupture. However, our findings do support a potential role of NGAL in the development of AAA