Effect and sustainability of a stepwise implemented multidisciplinary antimicrobial stewardship programme in a university hospital emergency department.

Objectives: To explore effectiveness and sustainability of guideline adherence and antibiotic consumption after establishing treatment guidelines and initiating antimicrobial stewardship (AMS) ward rounds in a university hospital emergency department (ED). Methods: Data were gathered retrospectively from 2017 to 2021 in the LMU University Hospital in Munich, Germany. Four time periods were compared: P1 (pre-intervention period); P2 (distribution of guideline pocket cards); P3 (reassessment after 3 years); and P4 (refresher of guideline pocket cards and additional daily AMS ward rounds for different medical disciplines). Primary outcome was adherence to guideline pocket cards for community-acquired pneumonia, cystitis, pyelonephritis and COVID-19-associated bacterial pneumonia. Secondary outcomes were reduction in antibiotic consumption and adherence to AMS specialist recommendations. Results: The study included 1324 patients. Guideline adherence increased in P2 for each of the infectious diseases entities. After 3 years (P3), guideline adherence decreased again, but was mostly on a higher level than in P1. AMS ward rounds resulted in an additional increase in guideline adherence (P1/P2: 47% versus 58.6%, P = 0.005; P2/P3: 58.6% versus 57.3%, P = 0.750; P3/P4: 57.3% versus 72.5%, P < 0.001). Adherence increased significantly, not only during workdays but also on weekends/nightshifts. Adherence to AMS specialist recommendations was excellent (91.3%). We observed an increase in use of narrow-spectrum antibiotics and a decrease in the application of fluoroquinolones and cephalosporins. Conclusions: Establishing treatment guidelines in the ED is effective. However, positive effects can be diminished over time. Daily AMS ward rounds are useful, not only to restore but to further increase guideline adherence significantly.

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 (MK) tracking and morphological analysis in vivo and in vitro, while not affecting lineage functions. Using Cre-driven Confetti expression, we provide insights into temporal gene expression patterns as well as spatial clustering of MK 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 wild-type controls. In conclusion, platelet lineage multicolor reporter mice prove useful in furthering our understanding of platelet and MK biology on a single cell level.

The AIM2 inflammasome exacerbates atherosclerosis in clonal haematopoiesis.

Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors1. Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2V617F (JAK2VF) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease1,2. Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1ß reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1ß or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.

Vascular surveillance by haptotactic blood platelets in inflammation and infection.

Breakdown of vascular barriers is a major complication of inflammatory diseases. Anucleate platelets form blood-clots during thrombosis, but also play a crucial role in inflammation. While spatio-temporal dynamics of clot formation are well characterized, the cell-biological mechanisms of platelet recruitment to inflammatory micro-environments remain incompletely understood. Here we identify Arp2/3-dependent lamellipodia formation as a prominent morphological feature of immune-responsive platelets. Platelets use lamellipodia to scan for fibrin(ogen) deposited on the inflamed vasculature and to directionally spread, to polarize and to govern haptotactic migration along gradients of the adhesive ligand. Platelet-specific abrogation of Arp2/3 interferes with haptotactic repositioning of platelets to microlesions, thus impairing vascular sealing and provoking inflammatory microbleeding. During infection, haptotaxis promotes capture of bacteria and prevents hematogenic dissemination, rendering platelets gate-keepers of the inflamed microvasculature. Consequently, these findings identify haptotaxis as a key effector function of immune-responsive platelets.

Endothelial CD40 Mediates Microvascular von Willebrand Factor-Dependent Platelet Adhesion Inducing Inflammatory Venothrombosis in ADAMTS13 Knockout Mice.

BACKGROUND: von Willebrand factor (vWF) plays an important role in platelet activation. CD40-CD40 ligand (CD40L) induced vWF release has been described in large vessels and cultured endothelium, but its role in the microcirculation is not known. Here, we studied whether CD40 is expressed in murine microvessels in vivo, whether CD40L induces platelet adhesion and leukocyte activation, and how deficiency of the vWF cleaving enzyme ADAMTS13 affects these processes. METHODS AND RESULTS: The role of CD40L in the formation of beaded platelet strings reflecting their adhesion to ultralarge vWF fibers (ULVWF) was analyzed in the murine cremaster microcirculation in vivo. Expression of CD40 and vWF was studied by immunohistochemistry in isolated and fixed cremasters. Microvascular CD40 was only expressed under inflammatory conditions and exclusively in venous endothelium. We demonstrate that CD40L treatment augmented the number of platelet strings, reflecting ULVWF multimer formation exclusively in venules and small veins. In ADAMTS13 knockout mice, the number of platelet strings further increased to a significant extent. As a consequence extensive thrombus formation was induced in venules of ADAMTS13 knockout mice. In addition, circulating leukocytes showed primary and rapid adherence to these platelet strings followed by preferential extravasation in these areas. CONCLUSION: CD40L is an important stimulus of microvascular endothelial ULVWF release, subsequent platelet string formation and leukocyte extravasation but only in venous vessels under inflammatory conditions. Here, the lack of ADAMTS13 leads to severe thrombus formation. The results identify CD40 expression and ADAMTS13 activity as important targets to prevent microvascular inflammatory thrombosis.

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 GPIbα-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.

Immune homeostasis and regulation of the interferon pathway require myeloid-derived Regnase-3.

The RNase Regnase-1 is a master RNA regulator in macrophages and T cells that degrades cellular and viral RNA upon NF-κB signaling. The roles of its family members, however, remain largely unknown. Here, we analyzed Regnase-3-deficient mice, which develop hypertrophic lymph nodes. We used various mice with immune cell-specific deletions of Regnase-3 to demonstrate that Regnase-3 acts specifically within myeloid cells. Regnase-3 deficiency systemically increased IFN signaling, which increased the proportion of immature B and innate immune cells, and suppressed follicle and germinal center formation. Expression analysis revealed that Regnase-3 and Regnase-1 share protein degradation pathways. Unlike Regnase-1, Regnase-3 expression is high specifically in macrophages and is transcriptionally controlled by IFN signaling. Although direct targets in macrophages remain unknown, Regnase-3 can bind, degrade, and regulate mRNAs, such as Zc3h12a (Regnase-1), in vitro. These data indicate that Regnase-3, like Regnase-1, is an RNase essential for immune homeostasis but has diverged as key regulator in the IFN pathway in macrophages.

Midkine drives cardiac inflammation by promoting neutrophil trafficking and NETosis in myocarditis.

Heart failure due to dilated cardiomyopathy is frequently caused by myocarditis. However, the pathogenesis of myocarditis remains incompletely understood. Here, we report the presence of neutrophil extracellular traps (NETs) in cardiac tissue of patients and mice with myocarditis. Inhibition of NET formation in experimental autoimmune myocarditis (EAM) of mice substantially reduces inflammation in the acute phase of the disease. Targeting the cytokine midkine (MK), which mediates NET formation in vitro, not only attenuates NET formation in vivo and the infiltration of polymorphonuclear neutrophils (PMNs) but also reduces fibrosis and preserves systolic function during EAM. Low-density lipoprotein receptor-related protein 1 (LRP1) acts as the functionally relevant receptor for MK-induced PMN recruitment as well as NET formation. In summary, NETosis substantially contributes to the pathogenesis of myocarditis and drives cardiac inflammation, probably via MK, which promotes PMN trafficking and NETosis. Thus, MK as well as NETs may represent novel therapeutic targets for the treatment of cardiac inflammation.

Cathelicidins prime platelets to mediate arterial thrombosis and tissue inflammation.

Leukocyte-released antimicrobial peptides contribute to pathogen elimination and activation of the immune system. Their role in thrombosis is incompletely understood. Here we show that the cathelicidin LL-37 is abundant in thrombi from patients with acute myocardial infarction. Its mouse homologue, CRAMP, is present in mouse arterial thrombi following vascular injury, and derives mainly from circulating neutrophils. Absence of hematopoietic CRAMP in bone marrow chimeric mice reduces platelet recruitment and thrombus formation. Both LL-37 and CRAMP induce platelet activation in vitro by involving glycoprotein VI receptor with downstream signaling through protein tyrosine kinases Src/Syk and phospholipase C. In addition to acute thrombosis, LL-37/CRAMP-dependent platelet activation fosters platelet-neutrophil interactions in other inflammatory conditions by modulating the recruitment and extravasation of neutrophils into tissues. Absence of CRAMP abrogates acid-induced lung injury, a mouse pneumonia model that is dependent on platelet-neutrophil interactions. We suggest that LL-37/CRAMP represents an important mediator of platelet activation and thrombo-inflammation.

HIF-1α Dependent Wound Healing Angiogenesis In Vivo Can Be Controlled by Site-Specific Lentiviral Magnetic Targeting of SHP-2.

Hypoxia promotes vascularization by stabilization and activation of the hypoxia inducible factor 1α (HIF-1α), which constitutes a target for angiogenic gene therapy. However, gene therapy is hampered by low gene delivery efficiency and non-specific side effects. Here, we developed a gene transfer technique based on magnetic targeting of magnetic nanoparticle-lentivirus (MNP-LV) complexes allowing site-directed gene delivery to individual wounds in the dorsal skin of mice. Using this technique, we were able to control HIF-1α dependent wound healing angiogenesis in vivo via site-specific modulation of the tyrosine phosphatase activity of SHP-2. We thus uncover a novel physiological role of SHP-2 in protecting HIF-1α from proteasomal degradation via a Src kinase dependent mechanism, resulting in HIF-1α DNA-binding and transcriptional activity in vitro and in vivo. Excitingly, using targeting of MNP-LV complexes, we achieved simultaneous expression of constitutively active as well as inactive SHP-2 mutant proteins in separate wounds in vivo and hereby specifically and locally controlled HIF-1α activity as well as the angiogenic wound healing response in vivo. Therefore, magnetically targeted lentiviral induced modulation of SHP-2 activity may be an attractive approach for controlling patho-physiological conditions relying on hypoxic vessel growth at specific sites.

The proteasome regulates collagen-induced platelet aggregation via nuclear-factor-kappa-B (NFĸB) activation.

INTRODUCTION: Platelets possess critical hemostatic functions in the system of thrombosis and hemostasis, which can be affected by a multitude of external factors. Previous research has shown that platelets have the capacity to synthesize proteins de novo and more recently a multicatalytic protein complex, the proteasome, has been discovered in platelets. Due to its vital function for cellular integrity, the proteasome has become a therapeutic target for anti-proliferative drug therapies in cancer. Clinically thrombocytopenia is a frequent side-effect, but the aggregatory function of platelets also appears to be affected. Little is known however about underlying regulatory mechanisms and functional aspects of proteasome inhibition on platelets. Our study aims to investigate the role of the proteasome in regulating collagen-induced platelet aggregation and its interaction with NFkB in this context. MATERIAL AND METHODS: Using fluorescence activity assays, platelet aggregometry and immunoblotting, we investigate regulatory interactions of the proteasome and Nuclear-factor-kappa-B (NFkB) in collagen-induced platelet aggregation. RESULTS: We show that collagen induces proteasome activation in platelets and collagen-induced platelet aggregation can be reduced with proteasome inhibition by the specific inhibitor epoxomicin. This effect does not depend on Rho-kinase/ROCK activation or thromboxane release, but rather depends on NFkB activation. Inhibition of the proteasome prevented cleavage of NFκB-inhibitor protein IκBα and decreased NFκB activity after collagen stimulation. Inhibition of the NFκB-pathway in return reduced collagen-induced platelet proteasome activity and cleavage of proteasome substrates. CONCLUSIONS: This work offers novel explanations how the proteasome influences collagen-dependent platelet aggregation by involving non-genomic functions of NFkB.

Survival protein anoctamin-6 controls multiple platelet responses including phospholipid scrambling, swelling, and protein cleavage.

Scott syndrome is a rare bleeding disorder, characterized by altered Ca(2+)-dependent platelet signaling with defective phosphatidylserine (PS) exposure and microparticle formation, and is linked to mutations in the ANO6 gene, encoding anoctamin (Ano)6. We investigated how the complex platelet phenotype of this syndrome is linked to defective expression of Anos or other ion channels. Mice were generated with heterozygous of homozygous deficiency in Ano6, Ano1, or Ca(2+)-dependent KCa3.1 Gardos channel. Platelets from these mice were extensively analyzed on molecular functions and compared with platelets from a patient with Scott syndrome. Deficiency in Ano1 or Gardos channel did not reduce platelet responses compared with control mice (P > 0.1). In 2 mouse strains, deficiency in Ano6 resulted in reduced viability with increased bleeding time to 28.6 min (control 6.4 min, P < 0.05). Platelets from the surviving Ano6-deficient mice resembled platelets from patients with Scott syndrome in: 1) normal collagen-induced aggregate formation (P > 0.05) with reduced PS exposure (-65 to 90%); 2) lowered Ca(2+)-dependent swelling (-80%) and membrane blebbing (-90%); 3) reduced calpain-dependent protein cleavage (-60%); and 4) moderately affected apoptosis-dependent PS exposure. In conclusion, mouse deficiency of Ano6 but not of other channels affects viability and phenocopies the complex changes in platelets from hemostatically impaired patients with Scott syndrome.

Dicer cleavage by calpain determines platelet microRNA levels and function in diabetes.

RATIONALE: MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus. OBJECTIVE: This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function. METHODS AND RESULTS: Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins. CONCLUSIONS: Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus.

The tyrosine phosphatase SHP-1 regulates hypoxia inducible factor-1α (HIF-1α) protein levels in endothelial cells under hypoxia.

INTRODUCTION: The tyrosine phosphatase SHP-1 negatively influences endothelial function, such as VEGF signaling and reactive oxygen species (ROS) formation, and has been shown to influence angiogenesis during tissue ischemia. In ischemic tissues, hypoxia induced angiogenesis is crucial for restoring oxygen supply. However, the exact mechanism how SHP-1 affects endothelial function during ischemia or hypoxia remains unclear. We performed in vitro endothelial cell culture experiments to characterize the role of SHP-1 during hypoxia. RESULTS: SHP-1 knock-down by specific antisense oligodesoxynucleotides (AS-Odn) increased cell growth as well as VEGF synthesis and secretion during 24 hours of hypoxia compared to control AS-Odn. This was prevented by HIF-1α inhibition (echinomycin and apigenin). SHP-1 knock-down as well as overexpression of a catalytically inactive SHP-1 (SHP-1 CS) further enhanced HIF-1α protein levels, whereas overexpression of a constitutively active SHP-1 (SHP-1 E74A) resulted in decreased HIF-1α levels during hypoxia, compared to wildtype SHP-1. Proteasome inhibition (MG132) returned HIF-1α levels to control or wildtype levels respectively in these cells. SHP-1 silencing did not alter HIF-1α mRNA levels. Finally, under hypoxic conditions SHP-1 knock-down enhanced intracellular endothelial reactive oxygen species (ROS) formation, as measured by oxidation of H2-DCF and DHE fluorescence. CONCLUSIONS: SHP-1 decreases half-life of HIF-1α under hypoxic conditions resulting in decreased cell growth due to diminished VEGF synthesis and secretion. The regulatory effect of SHP-1 on HIF-1α stability may be mediated by inhibition of endothelial ROS formation stabilizing HIF-1α protein. These findings highlight the importance of SHP-1 in hypoxic signaling and its potential as therapeutic target in ischemic diseases.

Hepatitis C virus induced endothelial inflammatory response depends on the functional expression of TNFα 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 (TNFα). HCV-RNA induces the endothelial expression of TNFα and TNFα 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.

The endothelial tyrosine phosphatase SHP-1 plays an important role for vascular haemostasis in TNFα -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 TNFα -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 TNFα -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 TNFα -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 TNFα -induced surface expression of p-selectin and von Willebrand factor. Additionally, TNFα 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 α -induced endothelial inflammation where it may serve as an autoinhibitory molecule to prevent excess inflammatory response and thrombus formation.

Combined deficiency in glutathione peroxidase 4 and vitamin E causes multiorgan thrombus formation and early death in mice.

RATIONALE: Growing evidence indicates that oxidative stress contributes markedly to endothelial dysfunction. The selenoenzyme glutathione peroxidase 4 (Gpx4) is an intracellular antioxidant enzyme important for the protection of membranes by its unique activity to reduce complex hydroperoxides in membrane bilayers and lipoprotein particles. Yet a role of Gpx4 in endothelial cell function has remained enigmatic. OBJECTIVE: To investigate the role of Gpx4 ablation and subsequent lipid peroxidation in the vascular compartment in vivo. METHODS AND RESULTS: Endothelium-specific deletion of Gpx4 had no obvious impact on normal vascular homeostasis, nor did it impair tumor-derived angiogenesis in mice maintained on a normal diet. In stark contrast, aortic explants from endothelium-specific Gpx4 knockout mice showed a markedly reduced number of endothelial branches in sprouting assays. To shed light onto this apparent discrepancy between the in vivo and ex vivo results, we depleted mice of a second antioxidant, vitamin E, which is normally absent under ex vivo conditions. Therefore, mice were fed a vitamin E-depleted diet for 6 weeks before endothelial deletion of Gpx4 was induced by 4-hydroxytamoxifen. Surprisingly, ≈80% of the knockout mice died. Histopathological analysis revealed detachment of endothelial cells from the basement membrane and endothelial cell death in multiple organs, which triggered thrombus formation. Thromboembolic events were the likely cause of various clinical pathologies, including heart failure, renal and splenic microinfarctions, and paraplegia. CONCLUSIONS: Here, we show for the first time that in the absence of Gpx4, sufficient vitamin E supplementation is crucial for endothelial viability.

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.

Hydrogen sulfide-releasing aspirin derivative ACS14 exerts strong antithrombotic effects in vitro and in vivo.

OBJECTIVE: Hydrogen sulfide (H(2)S)-releasing NSAIDs exert potent anti-inflammatory effects beyond classical cyclooxygenase inhibition. Here, we compared the platelet inhibitory effects of the H(2)S-releasing aspirin derivative ACS14 with its mother compound aspirin to analyze additional effects on platelets. METHODS AND RESULTS: In platelets of mice fed with ACS14 for 6 days (50 mg/kg per day), not only arachidonic acid-induced platelet aggregation but also ADP-dependent aggregation was decreased, an effect that was not observed with an equimolar dose of aspirin (23 mg/kg per day). ACS14 led to a significantly longer arterial occlusion time after light-dye-induced endothelial injury as well as decreased thrombus formation after ferric chloride-induced injury in the carotid artery. Bleeding time was not prolonged compared with animals treated with equimolar doses of aspirin. In vitro, in human whole blood, ACS14 (25-500 µmol/L) inhibited arachidonic acid-induced platelet aggregation, but compared with aspirin additionally reduced thrombin receptor-activating peptide-, ADP-, and collagen-dependent aggregation. In washed human platelets, ACS14 (500 µmol/L) attenuated αIIbß3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. CONCLUSIONS: The H(2)S-releasing aspirin derivative ACS14 exerts strong antiaggregatory effects by impairing the activation of the fibrinogen receptor by mechanisms involving increased intracellular cyclic nucleotides. These additional antithrombotic properties result in a more efficient inhibition of thrombus formation in vivo as achieved with aspirin alone.

Response of VEGF to activation of viral receptors and TNFα in human mesangial cells.

Vascular endothelial growth factor (VEGF) plays an important role in glomerular homeostasis as well as in the pathogenesis of kidney diseases as glomerulonephritis (GN) and diabetic nephropathy. Mesangial cells (MC), which are an integral part of the functional glomerular filtration barrier in that providing structural support, can behave like inflammatory cells and produce mediators as chemokines and growth factors; they are known to express viral receptors, with TLR3 having been attributed relevance in viral disease-associated GN. Experiments were performed on human MC in cell culture. Stimulation experiments were performed with poly (I:C) and hepatitis C RNA from patients with hepatitis C infection. We hereby show a TLR3-mediated upregulation of VEGF and its receptor subtype 2 (VEGF-R2) in human MC upon activation of viral receptors by poly (I:C) and hepatitis C virus. The increase in VEGF expression levels is further enhanced by tumor necrosis factor alpha (TNFα) which also induces the cytokines IL-6 and IL-8 as well as the chemokines MCP-1 and RANTES. These effects are potentiated by preincubation of MC with poly (I:C), just as the induction of the viral receptors TLR3, RIG-1, and MDA5 themselves. Moreover, MCP-1 itself is able to significantly increase mesangial VEGF expression. Therefore, with VEGF and VEGF-R2 being induced upon viral receptor activation in human MC, a novel role of TLR3 in mediating glomerular damage in virally induced or aggravated GN is inferred. TNFα and MCP-1 are seemingly important in amplifying VEGF effects in the setting of virally induced inflammation, with TNFα being also able to induce other mediators of glomerular pathology in GN.