The role of Src-homology 2 domain containing tyrosine phosphatase 2 in growth factor dependent endothelial signalling and angiogenesis

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in proliferation, survival and sprouting of human microvascular- and umbilical vein endothelial cells (HMEC, HUVEC) using antisense oligonucleotides (AS-ODN) and a pharmacological SHP-2 inhibitor (PtpI IV). Knock-down of SHP-2 decreased bFGF and PDGF dependent endothelial cell proliferation (p<0.01; n=12) as compared to nonsense oligonucleotide (NS-ODN) treatment. Cell cycle analysis by flow cytometric propidium iodide staining (p<0.01, n=6) and subsequent Annexin V staining (p<0.05, n=9) revealed a significantly higher number of apoptotic cells following SHP-2 AS-ODN transfection. Furthermore, inhibition of SHP-2 significantly impaired the formation of capillary like structures as well as new vessel sprouting in Matrigel embedded mouse aortic rings ex vivo. Finally, this was associated with a decreased phosphorylation of PI3-Kinase, Akt and ERK1/2. Our results indicate that SHP-2 promotes endothelial cell survival and proliferation, possibly by growth factor dependent PI3-K and MAP kinase activation, and is necessary for new vessel formation. These observations suggest SHP-2 to be a key enzyme in the control of angiogenesis

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

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-1β-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-1β-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

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\textless60~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 \geq7.5, 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 \geq7.5, 122 (36%) had documented liver or bile diagnoses. MELD 10-\textless15 (corresponding to CPS-B) was found for 105 (14%), MELD \geq15 (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

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

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

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

IS The GEF Cytohesin-2/ARNO Mediates Resistin induced Phenotypic Switching in Vascular Smooth Muscle Cells

The pro-inflammatory adipokine resistin induces a phenotypic switch of vascular smooth muscle cells (VSMC), a process decisive for atherosclerosis, including morphological changes, increased synthetic activity, proliferation and migration. The guanine-exchange factor ARNO (Cytohesin-2) has been shown to be important for morphological changes and migration of other cell types. In this study we dissected the role of ARNO in resistin induced VSMC phenotypic switching and signalling. Firstly, treatment with the cytohesin inhibitor Secin H3 prevented the resistin mediated induction of morphological changes in VSMC. Secondly, Secin H3 treatment as well as expression of an inactive ARNO (EK) reduced resistin induced VSMC synthetic activity, as assessed by matrix metalloproteinase 2 (MMP-2) expression, as well as the migration into a wound in vitro compared to ARNO WT expression. Thirdly, we found ARNO to influence MMP-2 expression and migration via activation of p38 MAPK and the JNK/AP-1 pathway. Interestingly, these processes were shown to be dependent on the binding of PIP3, as mutation of the ARNO PH-domain inhibited VSMC migration, MMP-2 expression as well as p38 MAPK and JNK signalling. Thus, we demonstrate that ARNO is an important link in resistin dependent cell signalling leading to morphological changes, MMP-2 production and migration of VSMC

Retrospective evaluation of the impact of non-oncologic chronic drug therapy on the survival in patients with bladder cancer

Background Chronic drug therapy may impact recurrence and survival of patients with bladder cancer and thus be of concern regarding drug choice and treatment decisions. Currently, data are conflicting for some drug classes and missing for others. Objective To analyze the impact of common non-oncologic chronic drug intake on survival in patients with bladder cancer and radical cystectomy. Setting. Patients with bladder cancer and radical cystectomy (2004-2018) at the University Hospital Munich. Method Data from an established internal database with patients with bladder cancer and radical cystectomy were included in a retrospective study. Drug therapy at the time of radical cystectomy and survival data were assessed and follow-up performed 3~months after radical cystectomy and yearly until death or present. Impact on survival was analyzed for antihypertensive, antidiabetic, anti-gout, antithrombotic drugs and statins, using the Kaplan-Meier method, log-rank test and Cox-regression models. Main outcome measure Recurrence free survival, cancer specific survival and overall survival for users versus non-users of predefined drug classes. Results Medication and survival data were available in 972 patients. Median follow-up time was 22~months (IQR 7-61). In the univariate analysis, a significant negative impact among users on recurrence free survival (n = 93; p = 0.038), cancer specific survival (n = 116; p < 0.001) and overall survival (n = 116; p < 0.001) was found for calcium-channel blockers, whereas angiotensin-receptor-blockers negatively influenced overall survival (n = 96; p = 0.020), but not recurrence free survival (n = 73; p = 0.696) and cancer specific survival (n = 96; p = 0.406). No effect of angiotensin-receptor-blockers and calcium-channel blockers was seen in the multivariate analysis. None of the other studied drugs had an impact on survival. Conclusion There was no impact on bladder cancer recurrence and survival for any of the analyzed drugs. Considering our results and the controverse findings in the literature, there is currently no evidence to withhold indicated drugs or choose specific drug classes among the evaluated non-oncologic chronic drug therapies. Thus, prospective studies are required for further insight. Trail registration This is part of the trial DRKS00017080, registered 11.10.2019

A new medication-based prediction score for postoperative delirium in surgical patients: development and proof of feasibility in a retrospective patient cohort

Structured risk screening for postoperative delirium (POD) considering prehospital medication is not established. We aimed to develop a POD-risk prediction score based on known risk factors and delirium-risk increasing drugs to be used by pharmacists during medication reconciliation at hospital admission, and to test for feasibility in a retrospective cohort of surgical patients. Therefore, established POD-risk factors and drugs were extracted from the literature and a score was generated. Following this, the score was tested for feasibility in a retrospective 3-month-cohort of surgical patients. For patients with higher scores suggesting higher probability of POD, patient charts were screened for documentation of POD. For development of the score, the following POD-risk factors were defined and points assigned for score calculation: age (≥65 years=1 point/≥75 years=2), male sex (1), renal insufficiency (RI; 1), hepatic impairment (HI; Model-of-endstage-liver-disease (MELD) 10-14=1/≥15=2), delirium-risk increasing drugs (1 point per drug class), anticholinergic drug burden (ACB; ≥3=1). In the retrospective test cohort of 1174 surgical patients these factors concerned: age ≥65 years 567 patients (48%)/≥75 years 303 (26%), male 652 (55%), RI 238 (20%), MELD 10-14 106 (9%)/≥15 65 (5%), ≥ 1 delirium-risk increasing drug 418 (36%), ACB ≥3 106 (9%). The median POD-risk prediction score was 2 (range 0-9). Of 146 patients (12%) with a score ≥ 5, POD was documented for 43 (30%), no evidence for POD for 91 (62%) and data inconclusive for 12 (8%). For scores of ≥ 7, POD was documented for 50% of the patients with sufficient POD documentation. Overall, POD documentation was poor. To summarize, we developed and successfully tested the feasibility of a POD-prediction-score assessable by pharmacists at medication reconciliation at hospital admission