Electron microscopic observation in case of platelet activation in a chronic haemodialysis subject

During haemodialysis (HD), platelets (PLTs) are activated and release granule contents. As HD treatment occurs three times a week, it has been demonstrated that PLTs are exhausted due to the repetitive character of the treatment. To identify PLT depletion morphologically, PLT evaluation was performed by light microscopy and electron microscopy (EM) in a chronic HD subject and a healthy reference subject. Blood samples were taken before the start of HD treatment for measurement of PLT count, PLT volume and size parameters. Blood smears were screened by light microscopy for qualitative evaluation of PLT granule containing cytoplasm, as indicated by its staining density. Morphological PLT parameters of surface area and size of dense bodies were assessed by EM. Data were compared with results of a group of 20 chronic HD subjects and a group of 20 healthy reference subjects. With respect to the percentage of PLTs with appropriate staining density (>75%), light microscopic evaluation showed that this value (9%) was within the range of a group of chronic HD subjects, but considerably below the reference range (70%). EM evaluation revealed an average PLT surface area and dense bodies area of respectively 42% and 31%, if the healthy reference subject was set on 100%. PLTs from a chronic HD subject are considerably smaller and substantially less granular than PLTs from a healthy reference subject. These findings support the hypothesis of PLT depletion in chronic HD subjects due to frequent PLT activation and/or increased urea concentrations

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

The F-BAR protein pacsin2 inhibits asymmetric VE-cadherin internalization from tensile adherens junctions

Vascular homoeostasis, development and disease critically depend on the regulation of endothelial cell-cell junctions. Here we uncover a new role for the F-BAR protein pacsin2 in the control of VE-cadherin-based endothelial adhesion. Pacsin2 concentrates at focal adherens junctions (FAJs) that are experiencing unbalanced actomyosin-based pulling. FAJs move in response to differences in local cytoskeletal geometry and pacsin2 is recruited consistently to the trailing end of fast-moving FAJs via a mechanism that requires an intact F-BAR domain. Photoconversion, photobleaching, immunofluorescence and super-resolution microscopy reveal polarized dynamics, and organization of junctional proteins between the front of FAJs and their trailing ends. Interestingly, pacsin2 recruitment inhibits internalization of the VE-cadherin complex from FAJ trailing ends and is important for endothelial monolayer integrity. Together, these findings reveal a novel junction protective mechanism during polarized trafficking of VE-cadherin, which supports barrier maintenance within dynamic endothelial tissue

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

α-B Crystallin Reverses High Diastolic Stiffness of Failing Human Cardiomyocytes

BACKGROUND: Cardiomyocytes with a less distensible titin and interstitial collagen contribute to the high diastolic stiffness of failing myocardium. Their relative contributions and mechanisms underlying loss of titin distensibility were assessed in failing human hearts. METHODS AND RESULTS: Left ventricular tissue was procured in patients with aortic stenosis (AS, n=9) and dilated cardiomyopathy (DCM, n=6). Explanted donor hearts (n=8) served as controls. Stretches were performed in myocardial strips, and an extraction protocol differentiated between passive tension (Fpassive) attributable to cardiomyocytes or to collagen. Fpassive-cardiomyocytes was higher in AS and DCM at shorter muscle lengths, whereas Fpassive-collagen was higher in AS at longer muscle lengths and in DCM at shorter and longer muscle lengths. Cardiomyocytes were stretched to investigate titin distensibility. Cardiomyocytes were incubated with alkaline phosphatase, subsequently reassessed after a period of prestretch and finally treated with the heat shock protein α-B crystallin. Alkaline phosphatase shifted the Fpassive-sarcomere length relation upward only in donor. Prestretch shifted the Fpassive-sarcomere length relation further upward in donor and upward in AS and DCM. α-B crystallin shifted the Fpassive-sarcomere length relation downward to baseline in donor and to lower than baseline in AS and DCM. In failing myocardium, confocal laser microscopy revealed α-B crystallin in subsarcolemmal aggresomes. CONCLUSIONS: High cardiomyocyte stiffness contributed to stiffness of failing human myocardium because of reduced titin distensibility. The latter resulted from an absent stiffness-lowering effect of baseline phosphorylation and from titin aggregation. High cardiomyocyte stiffness was corrected by α-B crystallin probably through relief of titin aggregation

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

Contractile arrest reveals calcium-dependent stimulation of SERCA2a mRNA expression in cultured ventricular cardiomyocytes

Objective: Downregulation of sarco-endoplasmic reticulum calcium ATPase 2a (SERCA2a) expression is a critical marker of pathological myocardial hypertrophy. The effects of calcium-dependent signaling and of contractile activity on the regulation of myocardial SERCA2a expression remain unclear. The present study dissociates effects of calcium-dependent signaling through calcineurin (CN) and calmodulin dependent protein kinase-II (CAMK-II), from effects of contractile activity in spontaneously contracting rat neonatal ventricular cardiomyocytes (NVCM) using 2,3-butanedione monoxime (BDM), which arrests contractions but maintains calcium fluxes. Methods: SERCA2a mRNA expression was analysed using Northern hybridisation in spontaneously contracting NVCM (control) and in NVCM treated with either BDM, L-type Ca 2+-channel blocker (verapamil), CN-blocker (cyclosporin A; CsA), CAMK-II blocker (KN-93), or combinations thereof. Transient transfection of the CN-dependent transcription factor nuclear factor of activated T-lymphocytes (NFATc), coupled to GFP, was used to detect NFAT nuclear translocation. The effects of CN/CAMK-II-dependent signaling were further dissected into effects of the transcription factors NFATc4 and myocyte enhancer factor 2c (MEF2c) on the activity of various SERCA2a promoter fragments using transient transfection assays. Results: Treatment with BDM induced a 2.5-fold rise in SERCA2a mRNA, which was abolished by addition of verapamil and was reduced by addition of CsA (-40%) and KN-93 (-20%). NFAT nuclear translocation was similar in control and BDM-treated NVCM. SERCA2a promoter activity was stimulated by NFATc4 and MEF2c, but only when both factors were co-transfected. Conclusion: Following contractile arrest with BDM, upregulation of SERCA2a mRNA expression by CN/CAMK-II signaling becomes evident. This upregulation is likely the result of synergistic stimulation of SERCA2a promoter activity by NFATc4 and MEF2c. Contractile activity opposes this upregulation through distinct and independent pathways