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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.Over a period of 3 months (02-04/2021), elective orthopaedic and trauma patients with eGFRDuring 53 working days, 712 (30.5%) of 2331 screened patients were included with an eGFRIn 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.

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.

Levosimendan is used in severe chronic cardiac insufficiency, also within the peri-operative setting. Real-life pharmacokinetic data in surgical patients is lacking, making therapeutic drug monitoring (TDM) of levosimendan, its pharmacologically active metabolite OR-1896, and its intermediate OR-1855 important. A simultaneous highly sensitive quantification of levosimendan and its metabolites in small-volume samples has not yet been described. Here, levosimendan (LLOQ 0.450 nM), OR-1896, and OR-1855 (LLOQ both 1.0 nM) were successfully quantified by LC-ESI-MS/MS after liquid-liquid extraction in 300 µL of blood. A short C8 column under reversed-phase conditions enabled simultaneous and fast quantification of levosimendan in the negative and the metabolites in the positive ionization mode in a single run within 2 min. Interestingly and unexpectedly, constitutional isomers of levosimendan metabolites with identical mass transitions and similar retention times were observed in surgical patients’ samples, which we identified as the metamizole metabolites 4-aminoantipyrine and 4-acetamidoantipyrine. A longer C8 column and a modified mobile phase enabled selective quantification of all analytes in a single run within 7 min. We developed, validated, and applied highly sensitive LC-ESI-MS/MS methods for simultaneous quantification of levosimendan and its metabolites, enabling efficient TDM of cardiac surgery patients even with additional metamizole administration.

Purpose Two to seven percent of the German adult population has a renal impairment (RI) with an estimated glomerular filtration rate (eGFR) 2. 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.

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.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; eGFR60 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.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.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.

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.

Background Hepatic insufficiency can affect patient safety and should therefore be considered during drug therapy. Hospital admission offers an ideal point to screen for patients at risk and to adjust drug therapy accordingly. Objective To assess the number of patients admitted to hospital with clinically elevated liver parameters. To identify high-risk patients in need of potential drug therapy adjustment to liver function by calculation of liver scores. Finally, to investigate whether pre-hospital medication needed adjustment to liver function. Setting Patients admitted to surgical wards of a tertiary teaching hospital. Method Surgical patients were included in a 3-month retrospective study. A pharmacist-led screening process, including recording of elevated liver parameters and calculation of liver scores (Child-Pugh-score, Model of End-stage Liver Disase [MELD], MELDNa), was used to assess frequency of hepatic insufficiency and patients potentially needing medication adjustment. Additionally, pre-hospital medication was checked for contraindications and correct dosage with regard to liver function. Main outcome measure Percentage of surgical patients with clinically elevated liver parameters at admission, percentage of patients with hepatic insufficiency potentially needing drug therapy adjustment, and percentage of pre-hospital drug intakes not adjusted to liver function. Results Of 1200 patients, 130 (11%) had at least one clinically relevant elevated liver parameter at hospital admission. Of these, need for drug adjustment to liver function was found for 16-36%, depending on the liver score used (equivalent to 2-4% of all patients), with the highest number of patients detected by the MELD- and MELDNa-score. Pre-hospital medication concerned 719 drug intakes and was contraindicated in 2%, dosage not adjusted in 3%, and evaluation not possible in 44% of all drug intakes due to lack of information on the drug. Conclusion A significant proportion of patients admitted for surgery have clinically elevated liver parameters and potentially need medication adjustment. A pharmacist-led screening already at hospital admission can support the identification of patients with clinically relevant elevated liver parameters and patients at risk by calculating liver scores under routine conditions. Evaluation of drug adjustment to liver function is challenging, since no data are available in routine resources for a considerable number of drugs.

Background: Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence. Methods: Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay. Findings: We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1β as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1β-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88. Interpretation: Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. Fund: German Research Foundation, LMU Mentoring excellence and FöFoLe Programme, Verein zur Förderung von Wissenschaft und Forschung, German Ministry of Education and Research. Keywords: Endothelial cells, IL-1β, MyD88, ROS, SHP-2, Sepsis

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 n = 116; p 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.

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.

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) in the tubular lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD displayed no chemotactic properties on neutrophils, did not directly activate β2 integrins and did not upregulate adhesion molecules on endothelial cells. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation in the renal interstitium with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies.

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.

Uromodulin (UMOD) is produced and secreted by tubular epithelial cells. Secreted UMOD polymerizes (pUMOD) within the lumen, where it regulates salt transport and protects the kidney from bacteria and stone formation. Under various pathological conditions, pUMOD accumulates within the tubular lumen and reaches extratubular sites where it may interact with renal interstitial cells. Here, we investigated the potential of extratubular pUMOD to act as a damage associated molecular pattern (DAMP) molecule thereby creating local inflammation. We found that intrascrotal and intraperitoneal injection of pUMOD induced leukocyte recruitment in vivo and led to TNF-α secretion by F4/80 positive macrophages. Additionally, pUMOD directly affected vascular permeability and increased neutrophil extravasation independent of macrophage-released TNF-α. Interestingly, pUMOD did not directly upregulate adhesion molecules on endothelial cells and did not directly activate β2 integrins on neutrophils. In obstructed neonatal murine kidneys, we observed extratubular UMOD accumulation with tubular atrophy and leukocyte infiltrates. Finally, we found extratubular UMOD deposits associated with peritubular leukocyte infiltration in kidneys from patients with inflammatory kidney diseases. Taken together, we identified extratubular pUMOD as a strong inducer of leukocyte recruitment, underlining its critical role in mounting an inflammatory response in various kidneys pathologies.

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.

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.

BACKGROUND: The endothelial glycocalyx plays a decisive role in maintaining vascular homeostasis. Previous animal models have mainly focused on in-vitro experiments or the isolated beating guinea pig heart. To further evaluate underlying mechanisms of up-and down regulation, knock-out animals seem to be a promising option. OBJECTIVE: Aim of the present study was to evaluate if an isolated mouse-heart model is suitable for glycocalyx research. METHODS: Isolated beating mouse hearts (C57/Bl6J) underwent warm, no-flow ischemia and successive reperfusion. Coronary effluent was analyzed by ELISA and Western blot for the glycocalyx core protein: syndecan-1. Hearts were prepared for either immunofluorescence or electron microscopy and lysed for Western blot analysis. RESULTS: An endothelial glycocalyx covering the total capillary circumference and syndecan-1 were detected by electron and immunofluorescence microscopy. Ischemia/reperfusion seriously deteriorated both findings. Confoundingly, syndecan-1 was not detectable either in the coronary effluent or in the lysates of blood-free hearts by ELISA or Western blot technique. CONCLUSIONS: Blood vessels of mouse hearts contain an endothelial glycocalyx comparable to that of other animals also with respect to its core protein syndecan-1. But, for studies including quantification of intravascular soluble glycocalyx constituents, the amount of syndecan-1 in mouse hearts seems to be too low.

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.

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 &alpha, 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.

Anti-angiogenic therapies are promising options for diseases with enhanced vessel formation such as tumors or retinopathies. In most cases, a site-specific local effect on vessel growth is required, while the current focus on systemic distribution of angiogenesis inhibitors may cause severe unwanted side-effects. Therefore, in the current study we have developed an approach for the local inhibition of vascularization, using complexes of lentivirus and magnetic nanoparticles in combination with magnetic fields. Using this strategy in the murine embryonic stem cell (ESC) system, we were able to site-specifically downregulate the protein tyrosine phosphatase SHP2 by RNAi technology in areas with active vessel formation. This resulted in a reduction of vessel development, as shown by reduced vascular tube length, branching points and vascular loops. The anti-angiogenic effect could also be recapitulated in the dorsal skinfold chamber of mice in vivo. Here, site-specific downregulation of SHP2 reduced re-vascularization after wound induction. Thus, we have developed a magnet-assisted, RNAi-based strategy for the efficient local inhibition of angiogenesis in ESCs in vitro and also in vivo.

Background Sepsis, the most severe form of infection, involves endothelial dysfunction which contributes to organ failure. To improve therapeutic prospects, elucidation of molecular mechanisms underlying endothelial vascular failure is of essence. Methods Polymicrobial contamination induced sepsis mouse model and primary endothelial cells incubated with sepsis serum were used to study SHP-2 in sepsis-induced endothelial inflammation. SHP-2 activity was assessed by dephosphorylation of pNPP, ROS production was measured by DCF oxidation and protein interactions were assessed by proximity ligation assay. Vascular inflammation was studied in the mouse cremaster model and in an in vitro flow assay. Findings We identified ROS-dependent inactivation of the tyrosine phosphatase SHP-2 to be decisive for endothelial activation in sepsis. Using in vivo and in vitro sepsis models, we observed a significant reduction of endothelial SHP-2 activity, accompanied by enhanced adhesion molecule expression. The impaired SHP-2 activity was restored by ROS inhibitors and an IL-1 receptor antagonist. SHP-2 activity inversely correlated with the adhesive phenotype of endothelial cells exposed to IL-1β as well as sepsis serum via p38 MAPK and NF-κB. In vivo, SHP-2 inhibition accelerated IL-1β-induced leukocyte adhesion, extravasation and vascular permeability. Mechanistically, SHP-2 directly interacts with the IL-1R1 adaptor protein MyD88 via its tyrosine 257, resulting in reduced binding of p85/PI3-K to MyD88. Interpretation Our data show that SHP-2 inactivation by ROS in sepsis releases a protective break, resulting in endothelial activation. Fund German Research Foundation, LMU Mentoring excellence and FoFoLe Programme, Verein zur Forderung von Wissenschaft und Forschung, German Ministry of Education and Research.

Hepatitis C virus (HCV) infection is a major problem worldwide. HCV is not limited to liver disease but is frequently complicated by immune-mediated extrahepatic manifestations such as glomerulonephritis or vasculitis. A fatal complication of HCV-associated vascular disease is thrombosis. Polyriboinosinic:polyribocytidylic acid (poly (I:C)), a synthetic analog of viral RNA, induces a Toll-like receptor 3 (TLR3)-dependent arteriolar thrombosis without significant thrombus formation in venules in vivo. These procoagulant effects are caused by increased endothelial synthesis of tissue factor and PAI-1 without platelet activation. In addition to human umbilical endothelial cells (HUVEC), human mesangial cells (HMC) produce procoagulatory factors, cytokines and adhesion molecules after stimulation with poly (I:C) or HCV-containing cryoprecipitates from a patient with a HCV infection as well. Activated protein C (APC) is able to prevent the induction of procoagulatory factors in HUVEC and HMC in vitro and blocks the effects of poly (I:C) and HCV-RNA on the expression of cytokines and adhesion molecules in HMC but not in HUVEC. In vivo, protein C inhibits poly (I:C)-induced arteriolar thrombosis. Thus, endothelial cells are de facto able to actively participate in immune-mediated vascular thrombosis caused by viral infections. Finally, we provide evidence for the ability of protein C to inhibit TLR3-mediated arteriolar thrombosis caused by HCV infection.

Connexin 43 (Cx43) expression is associated with an increased cell migration and related changes of the actin cytoskeleton (enhanced filopodia formation). These effects are mediated by the C-terminal cytoplasmic part of Cx43 in a channel-independent manner. Since this part has been shown to interact with a variety of proteins and has multiple phosphorylation sites we analyzed here a potential role of the protein kinase A (PKA) for the Cx43 mediated increase in cell migration. Mutation of the PKA-phosphorylation site (substitution of three serines by alanine or glycine) resulted in a further increase in cell motility compared to wild-type Cx43, but with a loss of directionality. Likewise, cell motility was enhanced by PKA inhibition only in Cx43 expressing cells, while reduced in the presence of the PKA activator forskolin. In contrast, cell motility remained unaffected by stimulation with forskolin in cells expressing Cx43 with the mutated PKA phosphorylation site (Cx43-PKA) as well as in Cx-deficient cells. Moreover, PKA activation resulted in increased binding of PKA and VASP to Cx43 associated with an enhanced phosphorylation of VASP, an important regulatory protein of cell polarity and directed migration. Functionally, we could confirm these results in endothelial cells endogenously expressing Cx43. A Tat-Cx43 peptide containing the PKA phosphorylation site abolished the PKA dependent reduction in endothelial cell migration. Our results indicate that PKA dependent phosphorylation of Cx43 modulates cell motility and plays a pivotal role in regulating directed cell migration.

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., Cathelicidins are antimicrobial peptides that eliminate pathogens and contribute to the innate immune response. Here the authors show that neutrophil-derived LL-37/CRAMP induces platelet activation and promotes arterial thrombosis and thrombo-inflammation.

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.

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.

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.

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 NF kappa B 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 (NF kappa B) 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 NF kappa B activation. Inhibition of the proteasome prevented cleavage of NF kappa B-inhibitor protein I kappa B alpha and decreased NF kappa B activity after collagen stimulation. Inhibition of the NF kappa B-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. (C) 2016 Elsevier Ltd. All rights reserved.

In the field of vascular gene therapy, targeting systems are promising advancements to improve site-specificity of gene delivery. Here, we studied whether incorporation of magnetic nanoparticles (MNP) with different magnetic properties into ultrasound sensitive microbubbles may represent an efficient way to enable gene targeting in the vascular system after systemic application. Thus, we associated novel silicon oxide-coated magnetic nanoparticle containing microbubbles (SO-Mag MMB) with lentiviral particles carrying therapeutic genes and determined their physico-chemical as well as biological properties compared to MMB coated with polyethylenimine-coated magnetic nanoparticles (PEI-Mag MMB). While there were no differences between both MMB types concerning size and lentivirus binding, SO-Mag MMB exhibited superior characteristics regarding magnetic moment, magnetizability as well as transduction efficiency under static and flow conditions in vitro. Focal disruption of lentiviral SO-Mag MMB by ultrasound within isolated vessels exposed to an external magnetic field decisively improved localized VEGF expression in aortic endothelium ex vivo and enhanced the angiogenic response. Using the same system in vivo, we achieved a highly effective, site-specific lentiviral transgene expression in microvessels of the mouse dorsal skin after arterial injection. Thus, we established a novel lentiviral MMB technique, which has great potential towards site-directed vascular gene therapy.

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 αIIbβ3 integrin activation and fibrinogen binding and increased intracellular cAMP levels and cAMP-dependent vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Conclusion— 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.

Site specific vascular gene delivery for therapeutic implications is favorable because of reduction of possible side effects. Yet this technology faces numerous hurdles that result in low transfection rates because of suboptimal delivery. Combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer could make it possible to use the systemic vasculature as the route of application and to magnetically trap these compounds at the target of interest. In this study we show that magnetic nanoparticle-coated microbubbles bind plasmid DNA and successfully deliver it to endothelial cells in vitro and more importantly transport their cargo through the vascular system and specifically deliver it to the vascular wall in vivo at sites where microbubbles are retained by magnetic force and burst by local ultrasound application. This resulted in a significant enhancement in site specific gene delivery compared with the conventional microbubble technique. Thus, this technology may have promising therapeutic potential. From the Clinical Editor This work focuses on combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer to enable targeted gene delivery via the systemic vasculature and magnetic trapping of these compounds at the target of interest.

Vascular endothelial cells, as well as smooth muscle cells, show heterogeneity with regard to their receptor expression and reactivity. For the vascular wall to act as a functional unit, the various cells' responses require integration. Such an integration is not only required for a homogeneous response of the vascular wall, but also for the vasomotor behaviour of consecutive segments of the microvascular arteriolar tree. As flow resistances of individual sections are connected in series, sections require synchronization and coordination to allow effective changes of conductivity and blood flow. A prerequisite for the local coordination of individual vascular cells and different sections of an arteriolar tree is intercellular communication. Connexins are involved in a dual manner in this coordination. (i) By forming gap junctions between cells, they allow an intercellular exchange of signalling molecules and electrical currents. In particular, the spread of electrical currents allows for coordination of cell responses over longer distances. (ii) Connexins are able to interact with other proteins to form signalling complexes. In this way, they can modulate and integrate individual cells' responses also in a channel-independent manner. This review outlines mechanisms allowing the vascular connexins to exert their coordinating function and to regulate the vasomotor reactions of blood vessels both locally, and in vascular networks. Wherever possible, we focus on the vasomotor behaviour of small vessels and arterioles which are the main vessels determining vascular resistance, blood pressure and local blood flow.

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.

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.

In glomerulonephritis, the migration of inflammatory cells into the glomerulus is an important step in disease initiation and progression. The viral receptor Toll-like receptor 3 (TLR3) is known to play a role in virus-associated glomerulonephritis. Based on this knowledge, this study aimed to define the effects of the TLR3 ligand polyriboinosinic:polyribocytidylic acid (poly(I:C)) on the expression of adhesion molecules and macrophage colony-stimulating factor (M-CSF) on resident glomerular cells. Experiments in MCs demonstrated that the activation of viral receptors by poly(I:C) leads to a time- and dose-dependent induction of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and M-CSF at both the mRNA and protein levels; these results were confirmed by incubating MCs with HCV RNA. As shown in knockdown experiments, this effect is specifically mediated by TLR3. The prestimulation of MCs with proinflammatory cytokines increases the effects of poly(I:C), except for its induction of VCAM-1. Tumor-necrosis factor (TNF)-α, likewise, induces ICAM-1, VCAM-1 and M-CSF, and amplifies the mesangial response to poly(I:C). These results were confirmed by incubating MCs with HCV RNA. We thus provide evidence that human MCs represent a potential target of the leukocytes and monocytes that infiltrate the glomerulus in viral disease-associated GN, highlighting the possibility that MCs may act as resident antigen-presenting 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.

Aims The vascular endothelial growth factor (VEGF) stimulates angiogenesis by induction of vessel permeability, proliferation, and migration of endothelial cells, an important process in ischaemic diseases. ADP-ribosylation factor (ARF) nucleotide-binding site opener (ARNO) (cytohesin-2) is a guanine exchange factor important for cellular signalling through ARF GTPases. However, a role for ARNO in VEGF-dependent endothelial processes has so far not been documented. Therefore, we investigated whether ARNO has a role in VEGF-dependent activation of endothelial cells and thus vessel permeability. Methods and results ARNO expression was observed in endothelial cells in vitro by RT–PCR, western blotting, and immunofluorescence as well as ex vivo by immunohistochemical staining of mouse aorta. Treatment with the cytohesin inhibitor SecinH3 or with an ARNO siRNA prevented VEGF-dependent Akt activation, assessed by detection of phosphorylated Akt, and proliferation of endothelial cells in vitro , measured by methylthiazoletetrazolium (MTT) reduction. In addition, ARNO suppression reduced VEGF-induced permeability in vessels of the mouse (C57BL/6) cremaster muscle in vivo , as measured by extravasation of fluorescein isothiocyanate (FITC)-dextran. Moreover, ARNO knock-down accelerated ligand-induced reduction in vascular endothelial growth factor receptor-2 (VEGFR-2) surface expression, internalization, and degradation, as assessed by flow cytometry and western blotting, respectively. Conclusion Our findings indicate an important and novel role for endothelial ARNO in VEGF-dependent initiation of angiogenesis by regulation of VEGFR-2 internalization in endothelial cells, resulting in the activation of the Akt pathway, vessel permeability, and ultimately endothelial proliferation. Thus, ARNO may be a new essential player in endothelial signalling and angiogenesis.

Cellular responses to the environment are mediated by intracellular signalling pathways monitoring several essential cellular processes, such as proliferation, migration, differentiation and survival. Cellular dysfunction is caused by dysregulation of intracelleular signalling pathways and may ultimately result in pathophysiological conditions. The non- transmembrane protein tyrosine phosphatase SHP-2 has been shown to be important for the control of cellular behaviour. It influences the activity of several growth factor and cytokine dependent signalling pathways by association with growth factor receptors, cell surface adhesion molecules and adaptor molecules such as Gab-1, Grb2 and IRS-1. Upon FGF-2, EGF and insulin stimulation SHP-2 regulates MAPK pathway activation. In addition, SHP-2 is involved in the regulation of cell survival by influencing the PI3-K/Akt pathway upon EGF, IGF and PDGF stimulation. Due to these properties, SHP-2 function has recently gained more interest in vascular processes, such as in the differentiation of cardiac progenitor cells and angiogenic events. Indeed, SHP-2 was shown to positively regulate endothelial cell motility and angiogenesis in vitro and in vivo as well as controlling intracellular pH of endothelial and vascular smooth muscle cells. On the other hand, SHP-2 was also demonstrated to be responsible for down regulation of VEGF receptor 2 activation upon dopamin and collagen stimulation. Finally, mutations in the Ptpn11 gene (encoding SHP-2) underlie the developmental disorders Noonan syndrome and Leopard syndrome characterized by congenital heart disease and hematologic abnormalities. Different mutations in this gene also result in myeloid and lymphoid malignancies. This article summarizes the role of SHP-2 in signalling pathways relevant for vascular biology and associated disorders.

Coating coronary stents with antirestenotic drugs revolutionized interventional cardiology. We developed a system for post-hoc drug delivery to uncoated stents. We coupled rapamycin or a chemically similar fluorescent dye to superparamagnetic nanoparticles. The antiproliferative activity of rapamycin coupled to nanoparticles was confirmed in vitro in primary porcine vascular cells. The particles were then incorporated into lipid based microbubbles. Commercially available stents were made magnetizable by nickel plating and used to induce strong field gradients in order to capture magnetic microbubbles from flowing liquids when placed in an external magnetic field. Nanoparticle bound Rapamycin dose dependently inhibited cell proliferation in vitro. Magnetic microcbubbles carrying coated nanoparticles were caught by magnets placed external to a flow-through tube. Plating commercial stents with nickel resulted in increased deposition at stent struts and allowed for widely increased distance of external magnets. Deposition depended on circulation time and velocity and distance of magnets. Deposited microbubbles were destroyed by ultrasound and delivered their cargo to targeted sites. Drugs can be incorporated into nanoparticle loaded microbubbles and thus be delivered to magnetizable stents from circulating fluids by applying external magnetic fields. This technology could allow for post-hoc drug coating of already implanted vascular stents.

Site specific vascular gene delivery is a promising tool for treatment of cardiovascular diseases. By combining ultrasound mediated microbubble destruction with site specific magnetic targeting of lentiviruses, we aimed to develop a technique suitable for systemic application.The magnetic nanoparticle coupling to lipid microbubbles was confirmed by absorbance measurements. Association of fluorescent lentivirus to magnetic microbubbles (MMB) was determined by microscopy and flow cytometry. Functionality and efficiency of GFP-encoding lentiviral MMB transduction was evaluated by endothelial (HMEC) GFP expression and cytotoxicity was measured by MTT reduction.Microbubbles with a mean diameter of 4.3 ± 0.04 μm were stable for 2 days, readily magnetizable and magnetically steerable in vitro and efficiently associated with lentivirus. Exposure of eGFP-encoding lentiviral MMB to human endothelial cells followed by application of an external static magnetic field (30 min) and ultrasonic destruction of the microbubbles did not markedly affect cellular viability. Finally, this combination led to a 30-fold increase in transduction efficiency compared to application of naked virus alone.By associating microbubbles with magnetic iron nanoparticles, these function as carriers for lentiviruses achieving tissue specific deposition at the site of interest.

Aims A CYP2C9-dependent endothelium-derived hyperpolarizing factor (EDHF) controls blood flow in many microvascular beds of various species by targeting vascular smooth muscle potassium channels. Since platelets express the same channels, we tested whether EDHF hyperpolarizes platelets and exerts an antithrombotic function in vivo . Methods and results Interaction of injected human platelets with the arteriolar wall (platelet-vessel wall interaction, PVWI) was assessed by intravital microscopy in skin muscle of awake hamsters. To understand the mechanisms of EDHF-induced platelet inhibition, we studied whether cultured human umbilical vein endothelial cells overexpressing CYP2C9-mRNA in vitro released a factor that could hyperpolarize human platelets. Under control conditions, there was no firm adhesion of platelets to the arteriolar wall, but temporary PVWI occurred. Local superfusion of the CYP2C9 inhibitor sulfaphenazole, at doses known to block EDHF-dependent dilations, significantly augmented PVWI, as did inhibition of NO synthase. Inhibition of both factors exerted additive effects on PVWI. Likewise, firm adhesion of a small fraction of platelets was observed. The prothrombotic effects of CYP2C9 inhibition in vivo were reversed by exogenous superfusion with 11,12-epoxyeicosatrienoic acids. Hyperpolarization reduced platelet adhesion to endothelial cells under static conditions in vitro and was dependent on calcium-activated potassium channels. The factor also reduced ADP-induced expression of platelet P-selectin, indicating reduction of platelet activity. Conclusion The arteriolar endothelium in vivo continuously releases a CYP2C9-derived EDHF. This EDHF exerts its effects by hyperpolarization of platelets through activation of KCa channels and reduction of platelet adhesion molecule expression, indicating that hyperpolarization reduces platelet activation. This demonstrates that EDHF is part of the antithrombotic properties of healthy endothelium in vivo .

Summary. Background: Diclofenac, like selective cyclooxygenase-2 inhibitors, which alter vascular levels of platelet active prostaglandins, has been reported to increase rates of acute myocardial infarction. Objective: The study was performed to investigate, in an animal model of arterial thrombosis in vivo, whether diclofenac differentially influences platelet activation and thrombosis in vessels under non-stimulated conditions or during acute systemic inflammation, such as induced by tumor necrosis factor-α (TNF-α). Methods: Platelet–vessel wall interaction (PVWI), firm platelet adhesion and arterial thrombosis following vessel injury were analyzed by intravital microscopy in arterioles of hamsters in the dorsal skinfold chamber model. Prostacyclin [prostaglandin I2 (PGI2)] and thromboxane A2 (TxA2) metabolites were measured. In vitro, endothelial adhesion molecule expression in cultured human microvascular endothelial cells was analyzed. Results: Under non-stimulated conditions, diclofenac (1 mg kg−1) enhanced PVWI, which was not mediated by increased adhesion molecule expression, but by decreased systemic PGI2 levels. Following ferric chloride-induced endothelial injury, diclofenac accelerated thrombotic vessel occlusion time, an effect that was reversed by the stable PGI2 analog iloprost. TNF-α, through induction of endothelial adhesion molecule expression, also enhanced PVWI, firm adhesion, and arterial thrombosis, but simultaneous treatment with TNF-α and diclofenac did not have an additive effect. Conclusions: By decreasing levels of PGI2 without, at the same time, altering prothrombotic TxA2 levels, diclofenac can exert prothrombotic effects. However, this is not the case when an inflammatory situation is created by TNF-α treatment. These data may explain the enhanced risk of acute myocardial infarction observed in patients taking diclofenac.

Angiogenesis initiation is crucially dependent on endothelial proliferation and can be stimulated by the fibroblast growth factor 2 (FGF-2). The DNA dependent protein kinase (DNA-PK), long known for its importance in repairing DNA double strand breaks, belongs to the phosphatidylinositol-3 kinase (PI3-K) super family and has recently been identified as one of the enzymes phosphorylating and activating Akt. Due to its similarity with PI3-K, we hypothesized that DNA-PK may have similar effects on endothelial angiogenic processes and signalling. We used primary endothelial cells (HUVEC and PAEC) and human microvascular endothelial cells (HMEC) to study the role of DNA-PK in endothelial proliferation and signalling. DNA-PKcs suppression with the compound NU7026 or with siRNA induced basal endothelial cell proliferation as well as enhanced FGF-2 dependent proliferation. This was associated with an increase in phosphorylated Akt. Tube formation was not affected by DNA-PKcs clearly showing that the role of DNA-PK in endothelial processes differs from that of PI3-K. Our findings indicate DNA-PK as an important enzyme maintaining the quiescent endothelial phenotype by actively inhibiting Akt thus restraining endothelial cell proliferation preventing excessive growth.

Endothelial cell survival is indispensable to maintain endothelial integrity and initiate new vessel formation. We investigated the role of SHP-2 in endothelial cell survival and angiogenesis in vitro as well as in vivo. SHP-2 function in cultured human umbilical vein and human dermal microvascular endothelial cells was inhibited by either silencing the protein expression with antisense-oligodesoxynucleotides or treatment with a pharmacological inhibitor (PtpI IV). SHP-2 inhibition impaired capillary-like structure formation (p < 0.01; n = 8) in vitroas well as new vessel growth ex vivo(p < 0.05; n = 10) and in vivo in the chicken chorioallantoic membrane (p < 0.01, n = 4). Additionally, SHP-2 knock-down abrogated fibroblast growth factor 2 (FGF-2)-dependent endothelial proliferation measured by MTT reduction (p < 0.01; n = 12). The inhibitory effect of SHP-2 knock-down on vessel growth was mediated by increased endothelial apoptosis (annexin V staining, p < 0.05, n = 9), which was associated with reduced FGF-2-induced phosphorylation of phosphatidylinositol 3-kinase (PI3-K), Akt and extracellular regulated kinase 1/2 (ERK1/2) and involved diminished ERK1/2 phosphorylation after PI3-K inhibition (n = 3). These results suggest that SHP-2 regulates endothelial cell survival through PI3-K-Akt and mitogen-activated protein kinase pathways thereby strongly affecting new vessel formation. Thus, SHP-2 exhibits a pivotal role in angiogenesis and may represent an interesting target for therapeutic approaches controlling vessel growth.

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.

Summary The body has the capacity to compensate for an occluded artery by creating a natural bypass upon increased fluid shear stress. How this mechanical force is translated into collateral artery growth (arteriogenesis) is unresolved. We show that extravasation of neutrophils mediated by the platelet receptor GPIbα and uPA results in Nox2-derived reactive oxygen radicals, which activate perivascular mast cells. These c-kit + /CXCR-4 + cells stimulate arteriogenesis by recruiting additional neutrophils as well as growth-promoting monocytes and T cells. Additionally, mast cells may directly contribute to vascular remodeling and vascular cell proliferation through increased MMP activity and by supplying growth-promoting factors. Boosting mast cell recruitment and activation effectively promotes arteriogenesis, thereby protecting tissue from severe ischemic damage. We thus find that perivascular mast cells are central regulators of shear stress-induced arteriogenesis by orchestrating leukocyte function and growth factor/cytokine release, thus providing a therapeutic target for treatment of vascular occlusive diseases.