Ferritin Levels on Hospital Admission Predict Hypoxic-Ischemic Encephalopathy in Patients After Out-of-Hospital Cardiac Arrest: A Prospective Observational Single-Center Study.

AIM: Out-of-hospital cardiac arrest (OHCA) is a major health concern in Western societies. Poor outcome after OHCA is determined by the extent of hypoxic-ischemic encephalopathy (HIE). Dysregulation of iron metabolism has prognostic relevance in patients with ischemic stroke and sepsis. The aim of this study was to determine whether serum iron parameters help to estimate outcomes after OHCA. METHODS: In this prospective single-center study, 70 adult OHCA patients were analyzed. Serum ferritin, iron, transferrin (TRF), and TRF saturation (TRFS) were measured in blood samples drawn on day 0 (admission), day 2, day 4, and 6 months after the return of spontaneous circulation (ROSC). The association of 4 iron parameters with in-hospital mortality, neurological outcome (cerebral performance category [CPC]), and HIE was investigated by receiver operating characteristics and multivariate regression analyses. RESULTS: OHCA subjects displayed significantly increased serum ferritin levels on day 0 and lowered iron, TRF, and TRFS on days 2 and 4 after ROSC, as compared to concentrations measured at a 6-month follow-up. Iron parameters were not associated with in-hospital mortality or neurological outcomes according to the CPC. Ferritin on admission was an independent predictor of features of HIE on cranial computed tomography and death due to HIE. CONCLUSION: OHCA is associated with alterations in iron metabolism that persist for several days after ROSC. Ferritin on admission can help to predict HIE.

A DARPin targeting activated Mac-1 is a novel diagnostic tool and potential anti-inflammatory agent in myocarditis, sepsis and myocardial infarction.

The monocyte ß2-integrin Mac-1 is crucial for leukocyte-endothelium interaction, rendering it an attractive therapeutic target for acute and chronic inflammation. Using phage display, a Designed-Ankyrin-Repeat-Protein (DARPin) was selected as a novel binding protein targeting and blocking the αM I-domain, an activation-specific epitope of Mac-1. This DARPin, named F7, specifically binds to activated Mac-1 on mouse and human monocytes as determined by flow cytometry. Homology modelling and docking studies defined distinct interaction sites which were verified by mutagenesis. Intravital microscopy showed reduced leukocyte-endothelium adhesion in mice treated with this DARPin. Using mouse models of sepsis, myocarditis and ischaemia/reperfusion injury, we demonstrate therapeutic anti-inflammatory effects. Finally, the activated Mac-1-specific DARPin is established as a tool to detect monocyte activation in patients receiving extra-corporeal membrane oxygenation, as well as suffering from sepsis and ST-elevation myocardial infarction. The activated Mac-1-specific DARPin F7 binds preferentially to activated monocytes, detects inflammation in critically ill patients, and inhibits monocyte and neutrophil function as an efficient new anti-inflammatory agent.

MicroRNA-100 Suppresses Chronic Vascular Inflammation by Stimulation of Endothelial Autophagy.

RATIONALE: The interaction of circulating cells within the vascular wall is a critical event in chronic inflammatory processes, such as atherosclerosis, but the control of the vascular inflammatory state is still largely unclear. OBJECTIVE: This study was undertaken to characterize the function of the endothelial-enriched microRNA miR-100 during vascular inflammation and atherogenesis. METHODS AND RESULTS: Based on a transcriptome analysis of endothelial cells after miR-100 overexpression, we identified miR-100 as a potent suppressor of endothelial adhesion molecule expression, resulting in attenuated leukocyte-endothelial interaction in vitro and in vivo as shown by flow cytometry and intravital imaging. Mechanistically, miR-100 directly repressed several components of mammalian target of rapamycin complex 1-signaling, including mammalian target of rapamycin and raptor, which resulted in a stimulation of endothelial autophagy and attenuated nuclear factor κB signaling in vitro and in vivo. In a low-density lipoprotein receptor-deficient atherosclerotic mouse model, pharmacological inhibition of miR-100 resulted in enhanced plaque lesion formation and a higher macrophage content of the plaque, whereas a systemic miR-100 replacement therapy had protective effects and attenuated atherogenesis, resulting in a decrease of plaque area by 45%. Finally, analysis of miR-100 expression in >70 samples obtained during carotid endarterectomy revealed that local miR-100 expression was inversely correlated with inflammatory cell content in patients. CONCLUSIONS: In summary, we describe an anti-inflammatory function of miR-100 in the vascular response to injury and inflammation and identify an important novel modulator of mammalian target of rapamycin signaling and autophagy in the vascular system. Our findings of miR-100 as a potential protective anti-athero-miR suggest that the therapeutic replacement of this microRNA could be a potential strategy for the treatment of chronic inflammatory diseases, such as atherosclerosis, in the future.

Chip-based digital PCR as a novel detection method for quantifying microRNAs in acute myocardial infarction patients.

miRNAs have shown promise as potential biomarkers for acute myocardial infarction (AMI). However, the current used quantitative real-time PCR (qRT-PCR) allows solely for relative expression of nucleic acids and it is susceptible to day-to-day variability, which has limited the validity of using the miRNAs as biomarkers. In this study we explored the technical qualities and diagnostic potential of a new technique, chip-based digital PCR, in quantifying the miRNAs in patients with AMI and ischaemia-reperfusion injury (I/R). In a dilution series of synthetic C.elegans-miR-39, chip-based digital PCR displayed a lower coefficient of variation (8.9% vs 46.3%) and a lower limit of detection (0.2 copies/µL vs 1.1 copies/µL) compared with qRT-PCR. In the serum collected from 24 patients with ST-elevation myocardial infarction (STEMI) and 20 patients with stable coronary artery disease (CAD) patients after percutaneous coronary intervention (PCI), we used qRT-PCR and multiplexed chip-based digital PCR to quantify the serum levels of miRNA-21 and miRNA-499 as they have been validated in AMI in prior studies. In STEMI, I/R injury was assessed via measurement of ST-segment resolution (ST-R). Chip-based digital PCR revealed a statistical significance in the difference of miR-21 levels between stable CAD and STEMI groups (118.8 copies/µL vs 59 copies/µL; P=0.0300), whereas qRT-PCR was unable to reach significance (136.4 copies/µL vs 122.8 copies/µL; P=0.2273). For miR-499 levels, both chip-based digital PCR and qRT-PCR revealed statistically significant differences between stable CAD and STEMI groups (2 copies/µL vs 8.5 copies/µL, P=0.0011; 0 copies/µL vs 19.4 copies/µL; P<0.0001). There was no association between miR-21/499 levels and ST-R post-PCI. Our results show that the chip-based digital PCR exhibits superior technical qualities and promises to be a superior method for quantifying miRNA levels in the circulation, which may become a more accurate and reproducible method for directly quantifying miRNAs, particularly for use in large multi-centre clinical trials.

MicroRNA-155 Exerts Cell-Specific Antiangiogenic but Proarteriogenic Effects During Adaptive Neovascularization.

BACKGROUND: Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice. METHODS AND RESULTS: Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155-deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155-deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1. CONCLUSIONS: Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow-derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice.

Inflammasome and toll-like receptor signaling in human monocytes after successful cardiopulmonary resuscitation.

BACKGROUND: Whole body ischemia-reperfusion injury (IRI) after cardiopulmonary resuscitation (CPR) induces a generalized inflammatory response which contributes to the development of post-cardiac arrest syndrome (PCAS). Recently, pattern recognition receptors (PRRs), such as toll-like receptors (TLRs) and inflammasomes, have been shown to mediate the inflammatory response in IRI. In this study we investigated monocyte PRR signaling and function in PCAS. METHODS: Blood samples were drawn in the first 12 hours, and at 24 and 48 hours following return of spontaneous circulation in 51 survivors after cardiac arrest. Monocyte mRNA levels of TLR2, TLR4, interleukin-1 receptor-associated kinase (IRAK)3, IRAK4, NLR family pyrin domain containing (NLRP)1, NLRP3, AIM2, PYCARD, CASP1, and IL1B were determined by real-time quantitative PCR. Ex vivo cytokine production in response to stimulation with TLR ligands Pam3CSK4 and lipopolysaccharide (LPS) was assessed in both whole blood and monocyte culture assays. Ex vivo cytokine production of peripheral blood mononuclear cells (PBMCs) from a healthy volunteer in response to stimulation with patients' sera with or without LPS was assessed. The results were compared to 19 hemodynamically stable patients with coronary artery disease. RESULTS: Monocyte TLR2, TLR4, IRAK3, IRAK4, NLRP3, PYCARD and IL1B were initially upregulated in patients following cardiac arrest. The NLRP1 and AIM2 inflammasomes were downregulated in resuscitated patients. There was a significant positive correlation between TLR2, TLR4, IRAK3 and IRAK4 expression and the degree of ischemia as assessed by serum lactate levels and the time until return of spontaneous circulation. Nonsurvivors at 30 days had significantly lower mRNA levels of TLR2, IRAK3, IRAK4, NLRP3 and CASP1 in the late phase following cardiac arrest. We observed reduced proinflammatory cytokine release in response to both TLR2 and TLR4 activation in whole blood and monocyte culture assays in patients after CPR. Sera from resuscitated patients attenuated the inflammatory response in cultured PBMCs after co-stimulation with LPS. CONCLUSIONS: Successful resuscitation from cardiac arrest results in changes in monocyte pattern recognition receptor signaling pathways, which may contribute to the post-cardiac arrest syndrome. TRIAL REGISTRATION: The trial was registered in the German Clinical Trials Register ( DRKS00009684 ) on 27/11/2015.

Antagonism and synergy between extracellular BMP modulators Tsg and BMPER balance blood vessel formation.

Growth and regeneration of blood vessels are crucial processes during embryonic development and in adult disease. Members of the bone morphogenetic protein (BMP) family are growth factors known to play a key role in vascular development. The BMP pathway is controlled by extracellular BMP modulators such as BMP endothelial cell precursor derived regulator (BMPER), which we reported previously acts proangiogenically on endothelial cells in a concentration-dependent manner. Here, we explore the function of other BMP modulators, especially Tsg, on endothelial cell behaviour and compare them to BMPER. In Matrigel assays, BMP modulators chordin and noggin had no stimulatory effect; however, gremlin and Tsg enhanced human umbilical vein endothelial cell (HUVEC) sprouting. As the activation dynamics of Tsg were similar to those of BMPER, we further investigated the proangiogenic effect of Tsg on endothelial cells. Tsg enhanced endothelial cell ingrowth in the mouse Matrigel plug assay as well as HUVEC sprouting, migration and proliferation in vitro, dependent on Akt, Erk and Smad signalling pathway activation in a concentration-dependent manner. Surprisingly, silencing of Tsg also increased HUVEC sprouting, migration and proliferation, which is again associated with Akt, Erk and Smad signalling pathway activation. Furthermore, we reveal that Tsg and BMPER interfere with each other to enhance proangiogenic events. However, in vivo the presence of Tsg as well as of BMPER is mandatory for regular development of the zebrafish vasculature. Taken together, our results suggest that BMPER and Tsg maintain a fine-tuned equilibrium that controls BMP pathway activity and is necessary for vascular cell homeostasis.

Selenium prevents microparticle-induced endothelial inflammation in patients after cardiopulmonary resuscitation.

INTRODUCTION: Microparticles are elevated in patients after successful cardiopulmonary resuscitation (CPR) and may play a role in the development of endothelial dysfunction seen in post-cardiac arrest syndrome (PCAS), a life threatening disease with high mortality. To identify mechanisms of endothelial activation and to develop novel approaches in the therapy of PCAS, the impact of selenium, a trace element with antioxidative properties, was characterized in endothelial dysfunction induced by microparticles of resuscitated patients. Additionally, course of plasma selenium levels was characterized in the first 72 hours post-CPR. METHODS: Endothelial cells were exposed to microparticles isolated of the peripheral blood of resuscitated patients, and leukocyte-endothelial interaction was measured by dynamic adhesion assay. Expression of adhesion molecules was assessed by immunoblotting and flow chamber. Blood samples were drawn 24, 48 and 72 hours after CPR for determination of plasma selenium levels in 77 resuscitated patients; these were compared to 50 healthy subjects and 50 patients with stable cardiac disease and correlated with severity of illness and outcome. RESULTS: Microparticles of resuscitated patients enhance monocyte-endothelial interaction by up-regulation of ICAM-1 and VCAM-1. Selenium administration diminished ICAM-1 and VCAM-1-mediated monocyte adhesion induced by microparticles of resuscitated patients, suggesting that selenium has anti-inflammatory effects after CPR. Lowered selenium plasma levels were observed in resuscitated patients compared to controls and selenium levels immediately and 24 hours after CPR, inversely correlated with clinical course and outcome after resuscitation. CONCLUSIONS: Endothelial dysfunction is a pivotal feature of PCAS and is partly driven by microparticles of resuscitated patients. Administration of selenium exerted anti-inflammatory effects and prevented microparticle-mediated endothelial dysfunction. Decline of selenium was observed in plasma of patients after CPR and is a novel predictive marker of ICU mortality, suggesting selenium consumption promotes inflammation in PCAS.

Inhibition of BMP activity protects epithelial barrier function in lung injury.

Epithelial injury is a central finding in pulmonary disease and is accompanied by disruption of epithelial barrier function, leading to pulmonary oedema and inflammation. Injured epithelial cells lose their properties and gain mesenchymal characteristics, a phenotypic switch that contributes to lung remodelling after injury. Here we studied bone morphogenetic protein (BMP) signalling and, in particular, the role of BMP2 and the BMP modulator BMPER in injured lung epithelium. Increased BMP activity, reflected by up-regulation of the Smad1/5-Id1 axis, is detected after injury of lung epithelium in vitro and in vivo. Two members of the BMP family, BMP2 and BMPER, have opposing effects. BMP2 is up-regulated after epithelial injury and causes epithelial dysfunction and hyperpermeability, mediated by the Smad1/5-Id1-dependent down-regulation of E-cadherin. In contrast, BMPER expression is decreased following injury, which in turn impairs epithelial integrity, characterized by reduction of E-cadherin and epithelial leakage in vitro and in vivo. High levels of BMPER antagonized BMP2-Smad5-Id1 signalling and prevented BMP2-mediated decrease of E-cadherin and hyperpermeability, suggesting that BMPER restores epithelial homeostasis. Supporting this notion, pharmacological inhibition of BMP signalling by LDN193189 prevented reduction of E-cadherin and disruption of epithelial barrier function. Inhibition of excessive BMP activation could be a new approach to restore epithelial integrity and prevent disruption of epithelial barrier function after lung injury.

Prognostic performance of the SCAI shock classification at admission and during ICU treatment: A retrospective, observational cohort study.

BACKGROUND: Cardiogenic shock (CS) is characterized by high mortality and requires accurate prognostic tools to predict outcomes and guide treatment. The Society for Cardiovascular Angiography and Interventions (SCAI) shock classification indicates shock severity and can be used for outcome prediction. OBJECTIVE: Here, we compare the prognostic performance of SCAI shock classification determined on admission and during intensive care unit (ICU) stay. METHODS: We included all patients with CS or conditions associated with developing CS based on ICD codes. SCAI shock stages were determined on admission and during the first 5 days of ICU stay. Receiver operating curves were used to compare the prognostic performance of SCAI stages on admission, SCAI stages during ICU stay and CS evolution (absent, resolved, persistent and new onset) for in-hospital mortality. RESULTS: Between 01/2018 and 06/2022, 1303 patients were identified and 862 patients were included. On admission, 50.6 % patients had SCAI shock stage A, 3.9 % SCAI shock stage B, 17.7 % SCAI shock stage C, 7.0 % SCAI shock stage D and 20.8 % SCAI shock stage E. Shock stage distribution changed dynamically during ICU stay. Compared to SCAI stage on admission (AUC 0.80; 95 % CI 0.77-0.83), highest achieved SCAI stage during ICU (AUC 0.86, 95 % CI 0.83-0.89, p < 0.0001) and shock evolution (AUC 0.87, 95 % CI 0.85-0.90, p < 0.0001) yielded better prognostic performance. CONCLUSIONS: SCAI shock stages changed dynamically during ICU stay, and prognostic performance can be improved by considering highest achieved SCAI shock stage as well as the evolution of CS compared to SCAI shock stage on admission.

BMP activity controlled by BMPER regulates the proinflammatory phenotype of endothelium.

The endothelium plays a pivotal role in vascular inflammation. Here we study bone morphogenetic protein (BMP) signaling in endothelial inflammation and in particular the role of BMPER, an extracellular BMP modulator that is important in vascular development and angiogenesis. Using the BMP antagonist dorsomorphin or BMP2 as an agonist we show that BMP signaling is essential for the inflammatory response of vascular endothelial cells as demonstrated by intravital microscopy. We found that BMPER is decreased in inflammation similar to vascular protective genes like KLF2 and eNOS. Using in vitro and in vivo models we show that BMPER is down-regulated through the TNFα-NFκB-KLF2 signaling pathway. Functionally, lack of BMPER induced by siRNA or in BMPER(+/-) mice confers a proinflammatory endothelial phenotype with reduced eNOS levels and enhanced expression of adhesion molecules leading to increased leukocyte adhesion and extravasation in ex vivo and in vivo experiments. Vice versa, addition of BMPER exerts endothelium protective functions and antagonizes TNFα induced inflammation. Mechanistically, we demonstrate that these effects of BMPER are dependent on BMP signaling because of enhanced NFκB activity. In conclusion, the BMP modulator BMPER is a new protective regulator of vascular inflammation that modulates leukocyte adhesion and migration in vitro and in vivo.

Bilateral deafness, diabetes, and different types of cardiomyopathy in family members with m.3243A > g mutation: a case report.

Background: The point mutation at position 3243 in the mitochondrial MT-TL1 gene (m.3243A > G) is a rare cause of hypertrophic cardiomyopathy (HCM). Information about HCM progression over time and occurrence of different cardiomyopathies in m.3243A > G carriers of the same family is still lacking. Case summary: A 48-year-old male patient was admitted to a tertiary care hospital with chest pain and dyspnoea. Bilateral hearing loss required hearing aids at the age of 40. A short PQ interval, narrow QRS complex, and inverted T-waves in lateral leads were present on the electrocardiogram. HbA1c of 7.3 mmol/L indicated prediabetes. Echocardiography excluded valvular heart disease and detected non-obstructive HCM with slightly reduced left ventricular ejection fraction (48%). Coronary artery disease was ruled out by coronary angiography. Myocardial fibrosis determined by repeated cardiac MRI progressed over time. Endomyocardial biopsy excluded storage disease, Fabry disease, and infiltrative and inflammatory cardiac disease. Genetic testing revealed m.3243A > G mutation in the MT-TL1 gene associated with mitochondrial disease. Clinical evaluation and genetic testing of the patients' family revealed five genotype-positive relatives with heterogeneous clinical phenotypes including deafness, diabetes mellitus, kidney disease, and both hypertrophic and dilated cardiomyopathy. Discussion: In patients with unexplained symmetric HCM with heterogenic clinical phenotypes at the organ levels, mitochondrial disease should be taken into consideration, particularly in the context of matrilinear transmission. m.3243A > G mutation is associated with mitochondrial disease in the index patient and five family members and leads to the diagnosis of maternally inherited diabetes and deafness with intra-familial variability of different cardiomyopathy forms.

MicroRNA-100 regulates neovascularization by suppression of mammalian target of rapamycin in endothelial and vascular smooth muscle cells.

BACKGROUND: The adaptive growth of blood vessels is an important protective mechanism in cardiovascular disease. However, the underlying regulatory mechanisms of this process are only partly understood. Recently, small endogenous RNAs (microRNAs [miRNAs]) were found to play an important role in embryonic and postnatal vascular development. Here, we used miRNA transcriptome analysis after induction of hind-limb ischemia in mice to screen for miRNAs involved in adaptive blood vessel growth following arterial occlusion. METHODS AND RESULTS: Using miRNA arrays, we explored the miRNA expression profile during adaptive neovascularization. We describe specific changes in miRNA expression patterns and show that miRNA-100 is significantly downregulated after induction of hind-limb ischemia in mice. Our data demonstrate that miR-100 modulates proliferation, tube formation, and sprouting activity of endothelial cells and migration of vascular smooth muscle cells and functions as an endogenous repressor of the serine/threonine protein kinase mammalian target of rapamycin (mTOR). Whereas miR-100 inhibition increased mTOR levels in endothelial cells, overexpression of miR-100 reduced mTOR expression and consequently attenuated cellular proliferation. Supporting this notion, overexpression of an mTOR construct lacking the miRNA binding site rescued the inhibitory effect of miR-100 on cell proliferation. Accordingly, miR-100 inhibition by specific antagomirs in vivo stimulated angiogenesis and resulted in functional improvement of perfusion after femoral artery occlusion in mice. In contrast, treatment with the mTOR inhibitor rapamycin had the opposite effect. CONCLUSIONS: Our data demonstrate that miR-100 has an antiangiogenic function and represses mTOR signaling in endothelial and vascular smooth muscle cells. Inhibition of miR-100 could be a novel approach for the modulation of blood vessel growth and other mTOR-dependent processes.

3D MRI provides improved visualization and detection of aortic arch plaques compared to transesophageal echocardiography.

PURPOSE: To compare 3D magnetic resonance imaging (3D MRI) with transesophageal echocardiography (TEE) for the detection of complex aortic plaques (≥4 mm thick, ulcerated, or containing mobile thrombi). MATERIALS AND METHODS: In all, 99 consecutive patients with acute cryptogenic stroke and ≥3 mm thick aortic plaques in TEE were prospectively included. 3D MRI comprised T1-weighted bright blood MRI with complete aortic coverage (spatial resolution 1 mm(3) ). Wall thickness and occurrence of complex plaques in ascending aorta (AAo), aortic arch (AA), and descending aorta (DAo) and image quality for each segment was rated for MRI and TEE. RESULTS: MRI detected more complex plaques than TEE (MRI vs. TEE): AAo 13 vs. 7; AA 37 vs. 11; differences were smaller in the DAo: 101 vs. 70. Image quality was higher for MRI in AAo and AA (P < 0.001) and superior for TEE in DAo (P < 0.001). MRI revealed additional complex plaques in the proximal aorta in 19 of 58 patients (32.8%) categorized as cryptogenic after complete routine diagnostics including TEE. CONCLUSION: Due to improved visualization of the aorta 3D MRI allows to detect more complex plaques than TEE. This renders 3D MRI particularly valuable for patients with cryptogenic stroke and for trials evaluating optimal treatment in aortic atherosclerosis.

BMPER is upregulated by statins and modulates endothelial inflammation by intercellular adhesion molecule-1.

OBJECTIVE: In addition to lowering cholesterol, statins exert pleiotropic effects on endothelial cells. Bone morphogenetic proteins (BMPs) have recently been implicated in vascular inflammation and disease. We set out to investigate the effect of statins on BMP endothelial cell precursor-derived regulator (BMPER), a novel member of the BMP pathway. METHODS AND RESULTS: Mevastatin enhanced BMPER expression in cultured endothelial cells in a time- and concentration-dependent manner as determined by immunocytochemistry, RT-PCR, and Western blotting. Similar effects were observed in vitro and in vivo using simvastatin. Actinomycin D chase analysis and BMPER promoter reporter assays revealed that this is mostly a posttranscriptional event resulting in prolonged BMPER RNA half-life. We confirmed that the RhoA/Rho-associated coiled-coil containing protein kinase Rho kinase (Rock)/actin pathway is involved using the specific pathway activator cytotoxic necrotizing factor of Yersinia pseudotuberculosis, which prevented upregulation of BMPER expression by mevastatin and pathway inhibitors (C3-toxin, RhoAN19 mutant, fasudil, and cytochalasin D) that enhanced BMPER expression. Increasing concentrations of BMPER exert antiinflammatory features in endothelial cells as reflected by intercellular adhesion molecule-1 downregulation. Accordingly, silencing of BMPER enhances intercellular adhesion molecule-1 expression. Furthermore, mevastatin reduced the expression of proinflammatory BMP4, a well-known direct interaction partner of BMPER. CONCLUSIONS: Mevastatin modulates the BMP pathway by enhancing BMPER via the RhoA/Rock/actin pathway, as well as by reducing BMP4 expression. BMP4 downregulation and BMPER upregulation contribute to the antiinflammatory pleiotropic effects of statins.

Circulating annexin V positive microparticles in patients after successful cardiopulmonary resuscitation.

INTRODUCTION: Ischemia/reperfusion after cardiopulmonary resuscitation (CPR) induces systemic inflammatory response and activation of endothelium and coagulation, resulting in a post-cardiac arrest syndrome. We analysed circulating (annexin V+) microparticles and their conjugates in resuscitated patients. METHODS: 36 patients after successful resuscitation, 20 control patients with stable cardiac disease and 15 healthy subjects were included prospectively. Two blood samples were drawn, one immediately and one 24 hours after return of spontaneous circulation (ROSC) to detect (annexin V+) monocyte-derived microparticles (MMPs) or procoagulant (annexin V+) platelet-derived microparticles (PMPs) and conjugates of endothelial-derived (annexin V+) microparticles (EMPs) with monocytes (EMP-MC) or platelets (EMP-PC). Measurements were performed by flow cytometric analysis. Additionally, the effect of isolated microparticles on cultured endothelial cells was assessed by ELISA. RESULTS: MMPs were significantly elevated immediately after ROSC compared to the cardiological control group (control; p < 0.01) and healthy subjects (healthy; p < 0.05) and persisted to be elevated in the following 24 hours after CPR (p < 0.05 vs. control and healthy, respectively). Procoagulant PMPs increased within the first 24 hours after ROSC (p < 0.01 vs. control and p < 0.005 vs. healthy). Conjugates of EMP with monocytes and platelets were both significantly elevated immediately after CPR (EMP-MC: p < 0.05 vs. control and p < 0.05 vs. healthy; EMP-PC: p < 0.05 vs. control and p < 0.05 vs. healthy), while only EMP-MC showed persisting high levels within 24 hours after CPR (p < 0.05 vs. control and p < 0.01 vs. healthy). MMP levels of ≥ 1.0/µL 24 hours after CPR predicted adverse outcome at 20 days (p < 0.05). Furthermore, isolated microparticles circulating in CPR patients early after ROSC led to enhanced endothelial apoptosis ex vivo compared to those of the healthy controls (p < 0.005). CONCLUSIONS: Resuscitated patients show substantially increased levels of different (annexin V+) microparticles and their conjugates immediately and 24 hours after cardiopulmonary resuscitation, suggesting an early onset of inflammation, an ongoing endothelial activation and a procoagulatory state. Additionally, microparticles of CPR patients may contribute to endothelial apoptosis. These results point to an involvement of microparticles in the development of the post-cardiac arrest syndrome.

Increased platelet, leukocyte and endothelial microparticles predict enhanced coagulation and vascular inflammation in pulmonary hypertension.

Pulmonary hypertension (PH) is associated with platelet activation, vascular inflammation and endothelial dysfunction leading to often life threatening thrombo-embolic complications. Microparticles (MPs) are cell vesicles with strong coagulatory and inflammatory effects being released during cell activation and apoptosis. As there are currently no established surrogate markers predicting platelet activation and pro-coagulation in PH patients, the aim of the study was to analyze different pro-coagulatory MP populations that might be related to thrombo-embolic complications in PH patients. Circulating MPs from platelet- (PMP, CD31(+)/61(+)), leukocyte- (LMP, CD11b(+)) and endothelial- (EMP, CD62E(+)) origin were measured by flow cytometry in 19 PH patients and were compared to 16 controls. PH patients had increased levels of PMP (PH vs. control 1,016 ± 201 vs. 527 ± 59 counts per min [cpm], P = 0.032), LMP (PH vs. control 31 ± 3 cpm vs. 18 ± 2 cpm, P = 0.001) and EMP (PH vs. control 99 ± 14 cpm vs. 46 ± 6 cpm, P = 0.001). Furthermore, PMP correlated to LMP (PMP vs. LMP: r = 0.75, P < 0.001) and LMP correlated to EMP levels (LMP vs. EMP, r = 0.74, P < 0.001) indicating a functional interaction between the different types of MP. In comparison to non-embolic PH patients, patients with a thrombo-embolic PH suffered from enhanced endothelial cell dysfunction as represented by significantly increased EMP levels (thrombo-embolic PH vs. non-embolic PH 137 ± 27 vs. 72 ± 10, P = 0.02). PH patients have increased levels of platelet-, leukocyte- and endothelial MP indicating an increased vascular pro-coagulation and inflammation which might be related to thrombo-embolic complications as well as PH progression.

Monocyte subset distribution and surface expression of HLA-DR and CD14 in patients after cardiopulmonary resuscitation.

Systemic inflammation is a major feature of the post-cardiac arrest syndrome. The three monocyte subpopulations are thought to play an important role in this inflammatory state because they are endowed with numerous pattern recognition receptors, such as CD14, that have been associated with ischemia-reperfusion injury. By contrast, an exaggerated antiinflammatory response has also been described following cardiac arrest, which may be mediated by downregulation of antigen presentation receptor HLA-DR. We report the composition of monocyte subpopulations and the expression of CD14 and HLA-DR following cardiac arrest. Blood specimens were collected from 32 patients at three timepoints in the first 48 h after cardiac arrest. Monocyte subset composition was determined by flow cytometry based on the expression of CD14, CD16, and HLA-DR. Monocyte subset composition and the expression of CD14 and HLA-DR were correlated with patient outcomes. The results were compared to 19 patients with coronary artery disease. Cardiac arrest patients showed a significant decline in the percentage of nonclassical monocytes. Monocyte CD14 expression was upregulated after 24 h and correlated with the time to return of spontaneous circulation. Downregulation of HLA-DR expression was observed mainly among classical monocytes and significantly correlated with the dose of norepinephrine used to treat shock. Downregulation of HLA-DR among nonclassical and intermediate monocytes was significantly associated with disease severity. Our data demonstrate the disturbance of monocyte subset composition with a significant decline in nonclassical monocytes at an early stage following cardiac arrest. Our findings suggest the simultaneous presence of hyperinflammation, as evidenced by upregulation of CD14, and monocyte deactivation, characterized by downregulation of HLA-DR. The extent of monocyte deactivation was significantly correlated with disease severity.

Complex plaques in the proximal descending aorta: an underestimated embolic source of stroke.

BACKGROUND AND PURPOSE: To investigate the incidence of retrograde flow from complex plaques (> or =4-mm-thick, ulcerated, or superimposed thrombi) of the descending aorta (DAo) and its potential role in embolic stroke. METHODS: Ninety-four consecutive acute stroke patients with aortic plaques > or =3-mm-thick in transesophageal echocardiography were prospectively included. MRI was performed to localize complex plaques and to measure time-resolved 3-dimensional blood flow within the aorta. Three-dimensional visualization was used to evaluate if diastolic retrograde flow connected plaque location with the outlet of the left subclavian artery, left common carotid artery, or brachiocephalic trunk. Complex DAo plaques were considered an embolic source if retrograde flow reached a supra-aortic vessel that supplied the territory of visible acute and embolic retinal or cerebral infarction. RESULTS: Only decreasing heart rate was correlated (P<0.02) with increasing flow reversal to the aortic arch. Retrograde flow from complex DAo plaques reached the left subclavian artery in 55 (58.5%), the left common carotid artery in 23 (24.5%), and the brachiocephalic trunk in 13 patients (13.8%). Based on routine diagnostics and MRI of the ascending aorta/aortic arch, stroke etiology was determined in 57 and cryptogenic in 37 patients. Potential embolization from DAo plaques was then identified in 19 of 57 patients (33.3%) with determined and in 9 of 37 patients (24.3%) with cryptogenic stroke. CONCLUSIONS: Retrograde flow from complex DAo plaques was frequent in both determined and cryptogenic stroke and could explain embolism to all brain territories. These findings suggest that complex DAo plaques should be considered a new source of stroke.