Platelet proteasome activity and metabolism is upregulated during bacterial sepsis

Dysregulation of platelet function can contribute to the disease progression in sepsis. The proteasome represents a critical and vital element of cellular protein metabolism in platelets and its proteolytic activity has been associated with platelet function. However, the role of the platelet proteasome as well as its response to infection under conditions of sepsis have not been studied so far. We measured platelet proteasome activity by fluorescent substrates, degradation of poly-ubiquitinated proteins and cleavage of the proteasome substrate Talin-1 in the presence of living E. coli strains and in platelets isolated from sepsis patients. Upregulation of the proteasome activator PA28 (PSME1) was assessed by quantitative real-time PCR in platelets from sepsis patients. We show that co-incubation of platelets with living E. coli (UTI89) results in increased degradation of poly-ubiquitinated proteins and cleavage of Talin-1 by the proteasome. Proteasome activity and cleavage of Talin-1 was significantly increased in α-hemolysin (HlyA)-positive E. coli strains. Supporting these findings, proteasome activity was also increased in platelets of patients with sepsis. Finally, the proteasome activator PA28 (PSME1) was upregulated in this group of patients. In this study we demonstrate for the first time that the proteasome in platelets is activated in the septic milieu

Perioptic Cerebrospinal Fluid Dynamics in Idiopathic Intracranial Hypertension

Purpose: To examine the cerebrospinal fluid (CSF) dynamics along the entire optic nerve in patients with idiopathic intracranial hypertension (IIH) and papilledema by computed tomographic (CT) cisternography.Methods: Retrospective analysis of CT cisternographies in 16 patients with a history of IIH and papilledema (14 females and 2 males, mean age: 49 ± 16 years). Contrast loaded CSF (CLCSF) was measured in Hounsfield Units (HU) at three defined regions of interest (ROI) along the optic nerve (orbital optic nerve portion: bulbar and mid-orbital segment, intracranial optic nerve portion) and additionally in the basal cistern. The density measurements in ROI 1, ROI 2, and ROI 3 consist of measurements of the optic nerve complex: optic nerve sheath, CLCSF filled SAS and optic nerve tissue. As controls served a group of patients (mean age: 60 ± 19 years) without elevated intracranial pressure and without papilledema.Results: In IIH patients the mean CLCSF density in the bulbar segment measured 65 ± 53 HU on the right and 63 ± 35 HU on the left side, in the mid-orbital segment 68 ± 37 HU right and 60 ± 21 HU left. In the intracranial optic nerve portion 303 ± 137 HU right and 323 ± 169 HU left and in the basal cistern 623 ± 188 HU. Within the optic nerve the difference of CLCSF density showed a highly statistical difference (p < 0.001) between the intracranial optic nerve portion and the mid-orbital segment. CLCSF density was statistically significantly (p < 0.001) reduced in both intraorbital optic nerve segments in patients with IIH compared to controls.Conclusions: The current study demonstrates reduced CLCSF density within the orbital optic nerve segments in patients with IIH and papilledema compared to 12 controls without elevated intracranial pressure and without papilledema. Impaired CSF dynamics could be involved in the pathophysiology of optic nerve damage in PE in IIH

The Endothelial Tyrosine Phosphatase SHP-1 Plays an Important Role for Vascular Haemostasis in TNF alpha-Induced Inflammation In Vivo

Introduction. Inflammation and endothelium-derived superoxides are important pathomechanisms in atherothrombotic diseases. We could previously show that the tyrosine phosphatase SHP-1 acts as a negative regulator in endothelial superoxide production. In this study we investigated the influence of SHP-1 on platelet-endothelium interaction and arterial thrombosis in TNF alpha-induced endothelial inflammation in vivo. Methods. Arteriolar thrombosis and platelet rolling in vivo were investigated in C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. Results. Inhibition of SHP-1 by the specific pharmacological inhibitor sodium stibogluconate did not significantly enhance platelet-endothelium interaction in vivo under physiological conditions but led to an augmented fraction of rolling platelets in TNF alpha-induced systemic inflammation. Accordingly, ferric-chloride-induced arteriolar thrombus formation, which was already increased by SHP-1 inhibition, was further enhanced in the setting of TNF alpha-induced inflammation. Platelet aggregation in vitro as well as ex vivo was not influenced by SHP-1-inhibition. In cultured endothelial cells, sodium stibogluconate increased TNF alpha-induced surface expression of p-selectin and von Willebrand factor. Additionally, TNF alpha increased SHP-1 activity and protein expression. Conclusions. The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular hemostasis in vivo, which is crucial in TNF alpha-induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation

Prothrombotic effects of tumor necrosis factor alpha in vivo are amplified by the absence of TNF-alpha receptor subtype 1 and require TNF-alpha receptor subtype 2

INTRODUCTION: Elevated serum levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) correlate with an increased risk for atherothrombotic events and TNFα is known to induce prothrombotic molecules in endothelial cells. Based on the preexisting evidence for the impact of TNFα in the pathogenesis of autoimmune disorders and their known association with an acquired hypercoagulability, we investigated the effects of TNFα and the role of the TNF receptor subtypes TNFR1 and TNFR2 for arteriolar thrombosis in vivo. METHODS: Arteriolar thrombosis and platelet-rolling in vivo were investigated in wildtype, TNFR1-/-, TNFR2-/- and TNFR1-/R2-/- C57BL/6 mice using intravital microscopy in the dorsal skinfold chamber microcirculation model. In vitro, expression of prothrombotic molecules was assessed in human endothelial cells by real-time PCR and flow cytometry. RESULTS: In wildtype mice, stimulation with TNFα significantly accelerated thrombotic vessel occlusion in vivo upon ferric chloride injury. Arteriolar thrombosis was much more pronounced in TNFR1-/- animals, where TNFα additionally led to increased platelet-endothelium-interaction. TNFα dependent prothrombotic effects were not observed in TNFR2-/- and TNFR1-/R2- mice. In vitro, stimulation of human platelet rich plasma with TNFα did not influence aggregation properties. In human endothelial cells, TNFα induced superoxide production, p-selectin, tissue factor and PAI-1, and suppressed thrombomodulin, resulting in an accelerated endothelial dependent blood clotting in vitro. Additionally, TNFα caused the release of soluble mediators by endothelial cells which induced prothrombotic and suppressed anticoagulant genes comparable to direct TNFα effects. CONCLUSIONS: TNFα accelerates thrombus formation in an in vivo model of arteriolar thrombosis. Its prothrombotic effects in vivo require TNFR2 and are partly compensated by TNFR1. In vitro studies indicate endothelial mechanisms to be responsible for prothrombotic TNFα effects. Our results support a more selective therapeutic approach in anticytokine therapy favouring TNFR2 specific antagonists

Hepatitis C Virus Induced Endothelial Inflammatory Response Depends on the Functional Expression of TNF alpha Receptor Subtype 2

In hepatitis C virus (HCV) infection, morbidity and mortality often result from extrahepatic disease manifestations. We provide evidence for a role of receptors of the innate immune system in virally induced inflammation of the endothelium in vitro and in vivo. Corresponding to the in vitro finding of an HCV-dependent induction of proinflammatory mediators in endothelial cells, mice treated with poly (I: C) exhibit a significant reduction in leukocyte rolling velocity, an increase in leukocyte adhesion to the vessel wall and an increased extravasation of leukocytes. HCV directly promotes activation, adhesion and infiltration of inflammatory cells into the vessel wall by activation of endothelial viral receptors. Poly (I: C) induces the expression of TLR3 in vivo and hereby allows for amplification of all of the aforementioned responses upon viral infection. Proinflammatory effects of viral RNA are specifically mediated by TLR3 and significantly enhanced by tumor necrosis factor alpha (TNFa). HCV-RNA induces the endothelial expression of TNFa and TNFa receptor subtype 2 and we provide evidence that leucocyte adhesion and transmigration in response to activation of viral RNA receptors seem to depend on expression of functional TNFR2. Our results demonstrate that endothelial cells actively participate in immune mediated vascular inflammation caused by viral infections

Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIb alpha-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies

Single platelet and megakaryocyte morpho-dynamics uncovered by multicolor reporter mouse strains in vitro and in vivo

Visualizing cell behavior and effector function on a single cell level has been crucial for understanding key aspects of mammalian biology. Due to their small size, large number and rapid recruitment into thrombi, there is a lack of data on fate and behavior of individual platelets in thrombosis and hemostasis. Here we report the use of platelet lineage restricted multi-color reporter mouse strains to delineate platelet function on a single cell level. We show that genetic labeling allows for single platelet and megakaryocyte tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Credriven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of megakaryocytes in the bone marrow. In the vasculature, shape analysis of activated platelets recruited to thrombi identifies ubiquitous filopodia formation with no evidence of lamellipodia formation. Single cell tracking in complex thrombi reveals prominent myosin-dependent motility of platelets and highlights thrombus formation as a highly dynamic process amenable to modification and intervention of the acto-myosin cytoskeleton. Platelet function assays combining flow cytrometry, as well as in vivo, ex vivo and in vitro imaging show unaltered platelet functions of multicolor reporter mice compared to WT controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and megakaryocyte biology on a single cell level