Feasibility of the MELD score as a screening tool for pharmacists to identify patients with impaired hepatic function at hospital admission.

WHAT IS KNOWN AND OBJECTIVE: Hepatic impairment (HI) is a known risk factor for drug safety. The MELD score (Model-for-endstage-liver-disease), calculated from serum creatinine, bilirubin and International Normalized Ratio (INR), is a promising screening tool corresponding to Child-Pugh Score (CPS) for drug adjustment. We tested the feasibility of MELD as an automatic screening tool accounting for correct calculation, interfering factors (IF) and detection of patients corresponding to CPS-B/C potentially requiring drug adjustment. METHODS: We retrospectively calculated MELD for a 3-month cohort of surgical patients and assessed need for adjustment of MELD parameters to standard values. IF for INR (oral anticoagulants) and serum creatinine (renal insufficiency (RI; eGFR<60 ml/min/1.73m²); as well as drugs elevating creatinine levels (DECL)) and the number of patients with MELD scores corresponding to CPS-B/C were analysed. For MELD ≥7.5, liver and bile diagnoses were recorded. RESULTS AND DISCUSSION: Of 1183 patients, MELD was calculable for 761 (64%; median 7.5, range 6.4-36.8). Parameters had to be adjusted for 690 (91%) patients. IF of parameters were RI in 172 (23%), INR-elevating drugs in 105 (14%) and DECL in 33 (4%) patients. Of 335 (44%) patients with MELD ≥7.5, 122 (36%) had documented liver or bile diagnoses. MELD 10-<15 (corresponding to CPS-B) was found for 105 (14%), MELD ≥15 (corresponding to CPS-C) for 66 (9%) of the 761 patients with a calculated MELD. Referred to all patients, drug adjustments due to possible HI were recommendable for 14% of patients with suspected CPS-B/C. WHAT IS NEW AND CONCLUSION: MELD is a feasible screening tool for HI as a risk factor for drug safety at hospital admission when appropriately considering correct parameter adjustment and RI and INR-elevating drugs as IF. Further evaluation of sensitivity and specificity is needed.

Benefit of medication reviews by renal pharmacists in the setting of a computerized physician order entry system with clinical decision support.

WHAT IS KNOWN AND OBJECTIVE: A 'renal pharmacist consultant service' (RPCS) reviewing patients' charts with renal impairment (RI) for drug-related problems (DRP) can foster patient safety. However, the benefit of this service in the new setting of a computerized physician order entry (CPOE)-system with a clinical decision support (CDS)-system is unknown. The aim of our study was to evaluate the general need for an RPCS on wards with a CPOE-CDS-system already in use and its effectiveness on prescription changes to ensure in-hospital patient safety. METHODS: Over a period of 3 months (02-04/2021), elective orthopaedic and trauma patients with eGFRabsolute /CrCl <60 ml/min at a German University Hospital received a medication review by a renal pharmacist for all medication entered into the CPOE-system (Meona®) by the treating physicians. Written consultations explaining identified DRP and recommending interventions to solve them, for example, dose or drug adaptation, were shared with the physicians directly in the drug chart tab of Meona®. In complex cases, DRP were additionally discussed via phone. The prescription changes were evaluated retrospectively. RESULTS AND DISCUSSION: During 53 working days, 712 (30.5%) of 2331 screened patients were included with an eGFRnon-indexed /CrCl <60 ml/min and a pharmacist-led medication review was performed for all medication presented in the CPOE-system (Meona®). In 79 of 712 (11.1%) patients, one or more DRP were detected (median 1 DRP (1-3) per patient) and written recommendations concerning 106 of 1090 (9.7%) drugs were shared via Meona®. In total, 104 DRP were identified, mostly caused by 'dosage too high' (n = 55, 52.9%), 'dosage regime wrong' (n = 13, 12.5%), and 'contraindication' (n = 9, 8.7%). Acceptance rate of recommendations was 74.0% (n = 77/104). In nine cases (8.7%), despite of specific recommendations, no adjustment of drugs was made because of lack of alternatives. In 11 (10.6%) cases, prescription remained unchanged for unknown reasons and in seven (6.7%) cases, the result was unknown due to discharge. WHAT IS NEW AND CONCLUSION: In the setting of prescribing in a CPOE-CDS-system, that provides physicians with advice for drug or dose adaption, the pharmacist-led medication reviews still identified DRP in orthopaedic and trauma patients with RI. A RPCS forwarding recommendations to solve DRP via the electronic medical record increased appropriate prescribing by physicians and, thus, may further improve patient safety.

Implementation of a renal pharmacist consultant service - Information sharing in paper versus digital form.

WHAT IS KNOWN AND OBJECTIVE: Renal impairment (RI) and renal drug-related problems (rDRP) often remain unrecognized in the community setting. A "renal pharmacist consultant service" (RPCS) at hospital admission can support patient safety by detecting rDRP. However, the efficient information sharing from pharmacists to physicians is still discussed. The aim of the study was to test the implementation of a RPCS and its effectiveness on prescription changes and to evaluate two ways of written information sharing with physicians. METHODS: Urological patients with eGFRnon-indexed of 15-59 ml/min and ≥1 drug were reviewed for manifest and potential rDRP at admission by a pharmacist. Written recommendations for dose or drug adaptation were forwarded to physicians comparing two routes: July-September 2017 paper form in handwritten chart; November 2017-January 2018 digital PDF document in the electronic patient information system and e-mail alert. Prescription changes regarding manifest rDRP were evaluated and compared with a previous retrospective study without RPCS. RESULTS AND DISCUSSION: The RPCS detected rDRP in 63 of 234 (26.9%) patients and prepared written recommendations (median 1 rDRP (1-5) per patient) concerning 110 of 538 (20.5%) drugs at admission. For manifest rDRP, acceptance rates of recommendations were 62.5% (paper) vs 42.9% (digital) (P = 0.16). Compared with the retrospective study without RPCS (prescription changes in 21/76 rDRP; 27.6%), correct prescribing concerning manifest rDRP significantly increased by 27.1%. WHAT IS NEW AND CONCLUSION: A RPCS identifies patients at risk for rDRP and significantly increases appropriate prescribing by physicians. In our hospital (no electronic order entry, electronic chart or ward pharmacists), consultations in paper form seem to be superior to a digital PDF document.

Cx43 Promotes Endothelial Cell Migration and Angiogenesis via the Tyrosine Phosphatase SHP-2.

The gap junction protein connexin 43 (Cx43) is associated with increased cell migration and to related changes of the actin cytoskeleton, which is mediated via its C-terminal cytoplasmic tail and is independent of its channel function. Cx43 has been shown to possess an angiogenic potential, however, the role of Cx43 in endothelial cell migration has not yet been investigated. Here, we found that the knock-down of Cx43 by siRNA in human microvascular endothelial cells (HMEC) reduces migration, as assessed by a wound assay in vitro and impaired aortic vessel sprouting ex vivo. Immunoprecipitation of Cx43 revealed an interaction with the tyrosine phosphatase SHP-2, which enhanced its phosphatase activity, as observed in Cx43 expressing HeLa cells compared to cells treated with an empty vector. Interestingly, the expression of a dominant negative substrate trapping mutant SHP-2 (CS) in HMEC, via lentiviral transduction, also impaired endothelial migration to a similar extent as Cx43 siRNA compared to SHP-2 WT. Moreover, the reduction in endothelial migration upon Cx43 siRNA could not be rescued by the introduction of a constitutively active SHP-2 construct (EA). Our data demonstrate that Cx43 and SHP-2 mediate endothelial cell migration, revealing a novel interaction between Cx43 and SHP-2, which is essential for this process.

Correct use of non-indexed eGFR for drug dosing and renal drug-related problems at hospital admission.

PURPOSE: Two to seven percent of the German adult population has a renal impairment (RI) with an estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73m2. This often remains unrecognized and adjustment of drug therapy is lacking. To determine renal function in clinical routine, the CKD-EPI equation is used to calculate an indexed eGFR (ml/min/1.73m2). For drug dosing, it has to be individualized to a non-indexed eGFR (ml/min) by the patient's body surface area. Here, we investigated the number of patients admitted to urological wards of a teaching hospital with RI between July and December 2016. Additionally, we correctly used the eGFRnon-indexed for drug and dosage adjustments and to analyse the use of renal risk drugs (RRD) and renal drug-related problems (rDRP). METHODS: In a retrospective observational study, urological patients with pharmacist-led medication reconciliation at hospital admission and eGFRindexed (CKD-EPI) of 15-59 ml/min/1.73m2 were identified. Indexed eGFR (ml/min/1.73m2) was recalculated with body surface area to non-indexed eGFR (ml/min) for correct drug dosing. Medication at admission was reviewed for RRD and based on the eGFRnon-indexed for rDRP, e.g. inappropriate dose or contraindication. RESULTS: Of 1320 screened patients, 270 (20.5%) presented with an eGFRindexed of 15-59 ml/min/1.73m2. After readjustment, 203 (15.4%) patients had an eGFRnon-indexed of 15-59 ml/min. Of these, 190 (93.6%) used ≥ 1 drugs at admission with 660 of 1209 (54.7%) drugs classified as RRD. At least one rDRP was identified in 115 (60.5%) patients concerning 264 (21.8%) drugs. CONCLUSION: Renal impairment is a common risk factor for medication safety in urologic patients admitted to a hospital. Considerable shifts were seen in eGFR-categories when correctly calculating eGFRnon-indexed for drug dosing purposes. The fact that more than half of the study patients showed rDRP at hospital admission underlines the need to consider this risk factor appropriately.

Extracellular Matrix-Specific Platelet Activation Leads to a Differential Translational Response and Protein De Novo Synthesis in Human Platelets.

Platelets are exposed to extracellular matrix (ECM) proteins like collagen and laminin and to fibrinogen during acute vascular events. However, beyond hemostasis, platelets have the important capacity to migrate on ECM surfaces, but the translational response of platelets to different extracellular matrix stimuli is still not fully characterized. Using 2D-gel electrophoresis, confocal microscopy, polysome analysis and protein sequencing by mass spectrometry, we demonstrate that platelets show a differential expression profile of newly synthesized proteins on laminin, collagen or fibrinogen. In this context, we observed a characteristic, ECM-dependent translocation phenotype of translation initiation factor eIF4E to the ribosomal site. eIF4E accumulated in polysomes with increased binding of mRNA and co-localization with vinculin, leading to de novo synthesis of important cytoskeletal regulator proteins. As the first study, we included a proteome analysis of laminin-adherent platelets and interestingly identified upregulation of essentially important proteins that mediate cytoskeletal regulation and mobility in platelets, such as filamin A, talin, vinculin, gelsolin, coronin or kindlin-3. In summary, we demonstrate that platelet activation with extracellular matrix proteins results in a distinct stimulus-specific translational response of platelets that will help to improve our understanding of the regulation of platelet mobility and migration.

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.

Heat-Shock Protein 27 (HSPB1) Is Upregulated and Phosphorylated in Human Platelets during ST-Elevation Myocardial Infarction.

Heat-shock proteins are a family of proteins which are upregulated in response to stress stimuli including inflammation, oxidative stress, or ischemia. Protective functions of heat-shock proteins have been studied in vascular disease models, and malfunction of heat-shock proteins is associated with vascular disease development. Heat-shock proteins however have not been investigated in human platelets during acute myocardial infarction ex vivo. Using two-dimensional electrophoresis and immunoblotting, we observed that heat-shock protein 27 (HSPB1) levels and phosphorylation are significantly increased in platelets of twelve patients with myocardial infarction compared to patients with nonischemic chest pain (6.4 ± 1.0-fold versus 1.0 ± 0.9-fold and 5.9 ± 1.8-fold versus 1.0 ± 0.8-fold; p < 0.05). HSP27 (HSPB1) showed a distinct and characteristic intracellular translocation from the cytoskeletal fraction into the membrane fraction of platelets during acute myocardial infarction that did not occur in the control group. In this study, we could demonstrate for the first time that HSP27 (HSPB1) is upregulated and phosphorylated in human platelets during myocardial infarction on a cellular level ex vivo with a characteristic intracellular translocation pattern. This HSP27 (HSPB1) phenotype in platelets could thus represent a measurable stress response in myocardial infarction and potentially other acute ischemic events.

The Phosphatase SHP-2 Activates HIF-1α in Wounds In Vivo by Inhibition of 26S Proteasome Activity.

Vascular remodeling and angiogenesis are required to improve the perfusion of ischemic tissues. The hypoxic environment, induced by ischemia, is a potent stimulus for hypoxia inducible factor 1α (HIF-1α) upregulation and activation, which induce pro-angiogenic gene expression. We previously showed that the tyrosine phosphatase SHP-2 drives hypoxia mediated HIF-1α upregulation via inhibition of the proteasomal pathway, resulting in revascularization of wounds in vivo. However, it is still unknown if SHP-2 mediates HIF-1α upregulation by affecting 26S proteasome activity and how the proteasome is regulated upon hypoxia. Using a reporter construct containing the oxygen-dependent degradation (ODD) domain of HIF-1α and a fluorogenic proteasome substrate in combination with SHP-2 mutant constructs, we show that SHP-2 inhibits the 26S proteasome activity in endothelial cells under hypoxic conditions in vitro via Src kinase/p38 mitogen-activated protein kinase (MAPK) signalling. Moreover, the simultaneous expression of constitutively active SHP-2 (E76A) and inactive SHP-2 (CS) in separate hypoxic wounds in the mice dorsal skin fold chamber by localized magnetic nanoparticle-assisted lentiviral transduction showed specific regulation of proteasome activity in vivo. Thus, we identified a new additional mechanism of SHP-2 mediated HIF-1α upregulation and proteasome activity, being functionally important for revascularization of wounds in vivo. SHP-2 may therefore constitute a potential novel therapeutic target for the induction of angiogenesis in ischemic vascular disease.

NO Augments Endothelial Reactivity by Reducing Myoendothelial Calcium Signal Spreading: A Novel Role for Cx37 (Connexin 37) and the Protein Tyrosine Phosphatase SHP-2.

OBJECTIVE: Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified. APPROACH AND RESULTS: Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction-dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one). CONCLUSIONS: Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.

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.

LL37 inhibits the inflammatory endothelial response induced by viral or endogenous DNA.

In viral infection, morbidity and mortality often result from extrahepatic disease manifestations such as vasculitis. We hereby show that human microvascular endothelial cells express viral receptors of the innate immune system which are induced upon ligand engagement. Furthermore, stimulation of endothelial cells with the synthetic analog of viral DNA, poly (dA:dT), human DNA and hepatitis B virus-containing immunoprecipitates from a patient with polyarteritis nodosa induces an inflammatory response including the upregulation of adhesion molecules, which is mediated exclusively by TLR9 and involves an IRF3-dependent pathway. Thus, endothelial cells are able to actively participate in immune mediated vascular inflammation caused by viral infections. Furthermore, we provide evidence for the ability of LL37 to bind and internalize viral or endogenous DNA into non-immune cells. DNA nucleotides internalized by LL37 suppress the production of proinflammatory mediators suggesting a protective effect against direct responses to viral infection or circulating DNA-fragments of endogenous origin.

Determining sensitivity and specificity of risk scores for QTc interval prolongation in hemato-oncology patients prescribed systemic antifungal therapy: a retrospective cross-sectional study.

BACKGROUND: QTc interval prolongation can result in potentially lethal arrhythmias. One risk factor is QTc-prolonging drugs, including some antifungals often used in hemato-oncology patients. Screening tools for patients at risk have not yet been investigated in this patient population. AIM: Our aim was to evaluate the sensitivity and specificity of five QTc risk scores in hemato-oncology patients receiving systemic antifungal therapy. METHOD: Data were retrieved from an internal study database including adult hemato-oncology patients prescribed systemic antifungal therapy. Data on QTc-prolonging medication, risk factors for QTc prolongation, and electrocardiograms (ECG) were collected retrospectively for a period of 12 months. The QTc risk scores according to Tisdale, Vandael, Berger, Bindraban, and Aboujaoude as well as their sensitivity and specificity were calculated. RESULTS: During the evaluated period, 77 patients were prescribed systemic antifungals resulting in 187 therapy episodes. Regarding therapy episodes, median age was 56 years (IQR 44-68), 41% (77) were female, and a median of 3 QTc-prolonging drugs were prescribed (range 0-6). ECGs were available for 45 (24%) of the therapy episodes 3-11 days after initiation of the antifungal therapy, 22 of which showed QTc prolongation. Regarding these 45 therapy episodes, sensitivity and specificity of the risk scores were calculated as follows: Tisdale 86%/22%, Vandael 91%/35%, Berger 32%/83%, Bindraban 50%/78%, Aboujaoude 14%/87%. CONCLUSION: The QTc risk scores according to Tisdale and Vandael showed sufficient sensitivity for risk stratification in the studied patient population. In contrast, risk scores according to Berger, Bindraban, and Aboujaoude cannot be considered suitable due to poor sensitivity.

Serum concentrations of levosimendan and its metabolites OR-1855 and OR-1896 in cardiac surgery patients with cardiopulmonary bypass.

Background: The inotropic drug levosimendan is often used as an individualized therapeutic approach perioperatively in cardiac surgery patients with cardiopulmonary bypass (CPB). Data regarding serum concentrations of levosimendan and its metabolites within this context is lacking. Methods: In this retrospective descriptive proof-of-concept study, total serum concentrations (TSC) and unbound fractions (UF) of levosimendan and its metabolites OR-1896 and OR-1855 in cardiac surgery patients with CPB were measured using LC-ESI-MS/MS. Simulation of expected levosimendan TSC was performed using Pharkin 4.0. Serum NT-proBNP was assessed with ELISA. Results: After levosimendan infusion (1.25 mg or 2.5 mg, respectively) after anaesthesia induction, a median TSC of 1.9 ng/ml and 10.4 ng/ml was determined in samples taken directly after surgery (T1). Median TSC of 7.6 ng/ml and 22.0 ng/ml, respectively, were simulated at T1. Whereas 1.1 ng/ml and 1.6 ng/ml TSC of OR-1896, respectively, was quantified the day after surgery (T2), TSC of the intermediate metabolite OR-1855 was mostly below the lower limit of quantification (LLOQ). The UF was 0.5% and 1.1% for levosimendan and 64.1% and 52.1% for OR-1896, respectively, with over half the samples being below LLOQ. NT-proBNP concentrations before surgery and T2 did not differ. Discussion: The low TSC, UF and unchanged NT-proBNP levels in combination with high variation of serum levels between patients suggest a need for optimized dosing regimen of levosimendan combined with therapeutic drug monitoring for such an individualized approach. In addition, the differences between the measured and estimated concentrations may suggest a possible influence of CPB on levosimendan serum concentrations.

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.

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.

The Effects of the Levosimendan Metabolites OR-1855 and OR-1896 on Endothelial Pro-Inflammatory Responses.

The calcium sensitizer levosimendan is used for the treatment of acute decompensated heart failure. A small portion (4-7%) of levosimendan is metabolized to the pharmacologically active metabolite OR-1896 via the inactive intermediate OR-1855. In addition, levosimendan has been shown to exert positive effects on the endothelium in vitro antagonizing vascular dysfunction and inflammation. However, the function of the levosimendan metabolites within this context is still unknown. In this study, we thus investigated the impact of the metabolites OR-1896 and OR-1855 on endothelial inflammatory processes in vitro. We observed a reduction of IL-1ß-dependent endothelial adhesion molecule ICAM-1 and VCAM-1 as well as interleukin (IL) -6 expression upon levosimendan treatment but not after treatment with OR-1855 or OR-1896, as assessed by western blotting, flow cytometry, and qRT-PCR. Instead, the metabolites impaired IL-1ß-induced ROS formation via inactivation of the MAPK p38, ERK1/2, and JNK. Our results suggest that the levosimendan metabolites OR-1896 and OR-1855 have certain anti-inflammatory properties, partly other than levosimendan. Importantly, they additionally show that the intermediate metabolite OR-1855 does, in fact, have pharmacological effects in the endothelium. This is interesting, as the metabolites are responsible for the long-term therapeutic effects of levosimendan, and heart failure is associated with vascular dysfunction and inflammation.

Hepatic Impairment as a Risk Factor for Drug Safety: Suitability and Comparison of Four Liver Scores as Screening Tools.

Hepatic impairment (HI) influences the pharmacokinetics and pharmacodynamics of drugs and represents an important risk factor for drug safety. A reliable screening tool for HI identification at hospital admission by pharmacists would be desirable but is currently lacking. Therefore, we tested four liver scores as potential screening instruments. We retrospectively recorded liver/bile diagnoses, symptoms and abnormalities (summarized as hepatic findings) of 200 surgical patients followed by an assessment of the relevance of these findings for drug therapy (rating). The agreement between the Model of Endstage Liver Disease (MELD), Non-alcoholic fatty liver disease fibrosis score (NFS), Fibrosis 4 index (FIB-4), and aspartate-aminotransferase to platelet ratio index (APRI) and the rating was quantified by Cohen's Kappa. The performance of the scores in this setting was further evaluated by their sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of 200 patients, 18 (9%) had hepatic findings relevant for drug therapy. Fair agreement was found for FIB-4 and MELD and slight agreement for APRI and NFS compared to the rating. The highest values for sensitivity, specificity, PPV, and NPV were 41.2% (MELD), 99.3% (APRI), 66.7% (APRI), and 93.6% (MELD), respectively. Due to low performance, none of the scores can be recommended for clinical use as a single screening tool for HI at hospital admission.

The Characterization of Non-oncologic Chronic Drug Therapy in Bladder Cancer Patients and the Impact on Recurrence-Free and Cancer-Specific Survival: A Prospective Study.

We aimed to characterize non-oncologic chronic drug therapy of bladder cancer (BC) patients and evaluate a possible impact on recurrence-free (RFS) and cancer-specific survival (CSS). Patients with a first diagnosis (FD) of BC or radical cystectomy (RC) were included in a prospective, monocentric, observational study. Drugs and medical data was assessed at start and three-monthly for 24 months. Drugs were classified by anatomical-therapeutic-chemical code (ATC). Endpoints for outcome analysis were RFS and CSS in univariate (Kaplan-Meier curves and log-rank test, Cox regression for Hazard Ratio (HR)) and multivariate (Cox regression models) analyses. Of 113 patients, 52 had FD and 78 RC. Median age was 74 and 72 years, 83% and 82% were male. Drugs of 114 ATC classes were taken by 48 (92%) FD patients (median number 4.5/IQR 2-7.5) and 73 (94%) of RC patients (median 5/IQR 2-9). In univariate analysis (log-rank test (p)/Cox regression (HR, 95% CI, p)), polypharmacy (p = 0.036/HR = 2.83, 95% CI = 1.02-7.90, p = 0.047), calcium channel blockers (p = 0.046/HR = 2.47, 95% CI = 0.97-6.27, p = 0.057) and proton pump inhibitors (p = 0.015/HR = 3.16, 95% CI = 1.18-8.41, p = 0.022) had a significant negative impact on RFS in RC patients, statins (p = 0.025/HR = 0.14, 95% CI = 0.02-1.06, p = 0.057) a positive effect on RFS in FD patients, angiotensin-converting enzyme inhibitors (p = 0.008/HR = 10.74, 95% CI = 1.20-96.17, p = 0.034) and magnesium (p = 0.042/HR = 5.28, 95% CI = 0.88-31.59, p = 0.067) a negative impact on CSS in FD patients. In multivariate analysis, the only significant drug effects were the negative impact of angiotensin-converting enzyme inhibitors (HR = 15.20, 95% CI = 1.30-177.67, p = 0.030) and magnesium (HR = 22.87, 95% CI = 1.57-333.81), p = 0.022) on CSS in FD patients, and the positive impact of statins (HR = 0.12, 95% CI = 0.01-0.97, p = 0.047) on RFS in FD patients. Impact of non-oncologic drugs on RFS and CSS was small in this prospective study. Thus, appropriate treatment of comorbidities is encouraged.

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.