A comparative study on adequate anesthesia depth: clinical judgement and the Narcotrend® measurement.

PURPOSE: To compare the clinical judgement of electroencephalogram (EEG)-naïve anesthesiologists with an EEG-based measurement of anesthetic depth (AD) using the Narcotrend® monitor. METHODS: In this prospective cohort study including 600 patients, AD during stable anesthesia was assessed by clinical judgement of the attending, EEG-blinded anesthesiologist (using a scale staging the AD as mid-adequate, adequate but fairly deep, or adequate but fairly light) and by simultaneously recorded Narcotrend measurements. RESULTS: In 42% of patients (n = 250), the anesthesiologist's clinical judgement was in agreement with anesthetic levels as measured by the Narcotrend monitor. In 46% of patients (n = 274), the anesthesiologist's judgement and the Narcotrend monitor differed by one AD level (minor discordance). Major discordance was observed in 76 (13%) measurements (judged deeper than measured, n = 29 [5%]; judged lighter than measured, n = 47 [8%]). In 7% of patients (n = 44), the Narcotrend index was outside the limits of adequate AD (too deep, n = 28 [5%]; too superficial, n = 16 [3%]). The overall level of agreement between the anesthesiologist's judgement and the Narcotrend monitor was not statistically significant (Cohen's kappa, -0.039; P = 0.17). Using a random forests algorithm, age, mean blood pressure, the American Society of Anesthesiologists classification, body mass index, and frailty were the variables with the highest relative feature importance to predict the level of agreement. CONCLUSION: These results suggest that clinical judgement of AD during stable anesthesia was not in agreement with EEG-based assessment of anesthetic depth in 58% of cases. Nevertheless, this finding could be influenced by the lack of validated scales to clinically judge AD. TRIAL REGISTRATION: www.clinicaltrials.gov (NCT02766894); registered 10 May, 2016.

RéSUMé: OBJECTIF: Notre objectif était de comparer le jugement clinique d'anesthésiologistes n'ayant pas accès à un électroencéphalogramme (EEG) à une mesure de la profondeur anesthésique (PA) fondée sur l'EEG utilisant le moniteur Narcotrend®. MéTHODE: Dans cette étude de cohorte prospective de 600 patients, la PA a été évaluée pendant la phase de maintien stable de l'anesthésie selon le jugement clinique de l'anesthésiologiste traitant, qui n'avait pas accès à l'EEG (sur une échelle évaluant la PA comme étant adéquate, adéquate mais relativement profonde ou adéquate mais relativement légère) et par des mesures simultanément enregistrées par le Narcotrend. RéSULTATS: Chez 42 % des patients (n = 250), le jugement clinique de l'anesthésiologiste concordait aux niveaux anesthésiques tels que mesurés par le moniteur Narcotrend. Chez 46 % des patients (n = 274), le jugement de l'anesthésiologiste et le moniteur Narcotrend différaient d'un niveau de PA (discordance mineure). Une discordance majeure a été observée dans 76 (13 %) mesures (jugées plus profondes que mesurées, n = 29 [5 %], jugées plus légères que mesurées, n = 47 [8 %]). Chez 7 % des patients (n = 44), l'indice Narcotrend était situé au-delà des limites d'une PA adéquate (trop profond, n = 28 [5 %]; trop superficiel, n = 16 [3 %]). Le niveau global de concordance entre le jugement de l'anesthésiologiste et le moniteur Narcotrend n'était pas significatif d'un point de vue statistique (kappa de Cohen, -0,039; P = 0,17). En se fondant sur un algorithme de forêt d'arbres décisionnels (random forests algorithm), l'âge, la tension artérielle moyenne, la classification selon l'American Society of Anesthesiologists, l'indice de masse corporelle et l'index de fragilité ont été identifiés comme les variables ayant la plus grande importance relative pour prédire le niveau de concordance. CONCLUSION: Ces résultats suggèrent que, dans 58 % des cas, le jugement clinique de la PA ne concordait pas à l'évaluation par EEG de la profondeur anesthésique pendant une phase de maintien stable de l'anesthésie. Toutefois, ces résultats pourraient être influencés par l'absence d'échelles validées pour juger la PA d'un point de vue clinique. ENREGISTREMENT DE L'éTUDE: www.clinicaltrials.gov (NCT02766894); enregistrée le 10 mai 2016.

Expression of the anti-inflammatory suppressor of cytokine signaling 3 (SOCS3) in human clear cell renal cell carcinoma.

The oncogenic transcription factor signal transducer and activator of transcription 3 (STAT3) is a cytokine-activated transcription factor controlling inflammation, cell proliferation, survival, and differentiation in normal tissue as well as in tumor growth. One of its most important negative regulators is the suppressor of cytokine signaling 3 (SOCS3). Here, we analyzed SOCS3 and other tumor-associated local immune regulators in human clear cell renal cell carcinoma (ccRCC). Analyses were performed in tumor and adjacent tumor-free healthy renal tissue from 35 patients with ccRCC. For functional analysis, ccRCC Caki-1 cell lines were stimulated with IL-6 and IFNγ in cell culture assays. We observed significantly lower SOCS3 messenger RNA (mRNA) levels in tumor tissue compared to healthy tissue. SOCS3 mRNA strongly correlated within tumor and healthy tissue. Interestingly vice versa, SOCS3 protein levels were significantly higher in tumor tissue than in healthy tissue. IL-22 and IL-22R1 mRNA displayed no differences in tumor and healthy tissue. Stimulation of Caki-1 cells with IFNγ resulted in markedly increased SOCS3 mRNA levels. We conclude that SOCS3 along with STAT3 participates in regulatory mechanisms in ccRCC, which certainly features only one of multiple factors involved but nevertheless merits further attention.

Combined peri-ischemic administration of Bß15-42 in treating ischemia reperfusion injury of the mouse kidney.

The disruption of endothelial integrity is a crucial step for the development of vascular leakage and consequently ischemia-reperfusion injury (IRI). Regarding the molecular cell-cell interaction, the fibrinopeptide Bß15-42 prevents vascular leakage by stabilizing the inter-endothelial junctions via association with the vascular endothelial-cadherin. In a previous study we showed that a renoprotective effect in early IRI may be achieved by intravenous administration of Bß15-42 at the time of reperfusion. We now aimed to investigate whether additional pre-ischemic application of Bß15-42 could enhance this effect. Therefore C57BL/6 mice were subjected to 0.5h bilateral renal ischemia followed by reperfusion. The animals were randomized into 6 groups (n=6): two control groups treated with i.v. administration of NaCl at reperfusion for 0.5h (NaCl 1h) and 2.5h (NaCl 3h), two groups with Bß15-42 at reperfusion for 0.5h (Bß(rep) 1h) and 2.5h (Bß(rep) 3h), and two groups with administration of Bß15-42 immediately pre-ischemic as well as at reperfusion for 0.5h (Bß(peri) 1h) and 2.5h (Bß(peri) 3h). We found that both Bß(rep) and Bß(peri) mice displayed reduced early renal damage compared with NaCl treated mice. However, there was no further reduction of the IR damage through added pre-ischemic application of Bß15-42. Overall, we detected significantly reduced endothelial activation, lower tissue infiltration of neutrophils as well as lower tissue levels of neutrophil gelatinase-associated lipocalin (NGAL) in all mice treated with Bß15-42 compared to mice treated with NaCl. Our data confirm the renoprotective effect of Bß15-42 in the early therapeutic treatment of acute kidney injury due to ischemia and reperfusion. However, a combined pre-and post-ischemic administration of Bß15-42 appears to provide no additional benefit compared with a sole administration at reperfusion.

Serum and urinary NGAL but not KIM-1 raises in human postrenal AKI.

BACKGROUND: We examined the value of the novel acute kidney injury (AKI) markers neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1) in acute postrenal impairment. These biomarkers have been evaluated in prerenal and intrarenal AKI so far, but not in human acute postrenal kidney injury. With regard to multimorbid and critically ill patients the discrimination of different AKI origins often remains a challenge. As the trend goes towards a diagnostic panel of AKI markers, we hereby aim to contribute to evaluate further options of discrimination in an observational case-control study. MATERIALS AND METHODS: Blood and urine samples were obtained from 53 patients with acute obstructive nephropathy secondary to ureteral calculi and 52 age-matched healthy controls. Serum NGAL (sNGAL), urinary NGAL (uNGAL) and urinary KIM-1 (uKIM-1) levels were determined using a commercially available ELISA kit, creatinine applying the Jaffé's method. RESULTS: While urinary levels of KIM-1 were not significantly different between patients with obstructive nephropathy and controls, a striking increase in sNGAL (P < 0·001) and uNGAL (P < 0·01) levels was detected in the obstructive nephropathy group. Within the obstructive nephropathy group, sNGAL (P = 0·01) and uNGAL (P = 0·049) but not uKIM-1 correlated positively with the white blood cell count and uNGAL correlated positively (P = 0·002) with the extent of leucocyturia. CONCLUSIONS: High levels of sNGAL and uNGAL observed in stone-induced acute obstructive nephropathy may represent a valuable marker of postrenal AKI. Low uKIM-1 levels may help to discriminate postrenal AKI events using a panel of markers in this setting.

Vitamin D hydroxylases CYP2R1, CYP27B1 and CYP24A1 in renal cell carcinoma.

BACKGROUND: There is increasing evidence that vitamin D metabolites influence carcinogenesis. Besides its role in mineral homoeostasis, calcitriol, the active metabolite of vitamin D (1,25(OH)2 D3 ), is known to possess antiproliferative, proapoptotic and immunomodulatory effects in cancer. Concerning the synthesis of vitamin D, the hydroxylases CYP2R1, CYP27B1 and CYP24A1 play a critical role, and the latter molecule determines the biological half-life of 1,25(OH)2 D3 , which is synthesized in the proximal renal tubules. MATERIALS AND METHODS: The adjacency of these two biological processes prompted us to investigate the gene expression of CYP2R1, CYP27B1 and CYP24A1 in patients with ccRCC. Using RT-PCR, we retrospectively compared mRNA expression profiles from human ccRCC tumour samples with those derived from the corresponding adjacent healthy tissues (n = 30). RESULTS: We observed that all three genes (CYP2R1, CYP27B1 and CYP24A1) were upregulated in tumours compared with normal tissue (P < 0·0001). Moreover, CYP24A1 displayed a significantly higher expression in tumours than CYP27B1 (P < 0·05) and CYP2R1 (P < 0·0001), whereas no differences in the expression of these genes were found in healthy renal tissue. Gene expression of CYP2R1, CYP27B1 and CYP24A did not differ between pathological classifications (TNM, grading, presence of metastasis). CONCLUSION: We thus conclude that upregulated gene expression of the catabolizing CYP24A1 as well as the synthesizing CYP2R1 and CYP27B1 may lead to a misbalance of vitamin D metabolites in ccRCC and thus contributing to its pathogenesis.

Cecum ligation and dissection: a novel modified mouse sepsis model.

BACKGROUND: The fact that many sepsis therapeutics failed to be translated into the human indicates that there is still a serious need to reassess our models of sepsis research. We aimed to develop a novel modified model of sepsis in the mouse, which simulates the clinical situation more accurately. MATERIALS AND METHODS: Sepsis was induced in C57Bl/6 mice by dissecting the cecum and placing the discontinued organ back into the abdomen (cecum ligation and dissection [CLD]). Septic animals were relaparotomized after 6 h, followed by peritoneal lavage, and antibiotic treatment. Results were compared with shams or the classic colon ligation and puncture (CLP) model. The postoperative lung impairment was assessed using neutrophil invasion as a surrogate. Proinflammatory cytokines were measured by either real-time polymerase chain reaction or Luminex technology, and liver damage was evaluated by aspartate transaminase and alanine transaminase measurements. RESULTS: In CLD animals with relaparotomy after 6 h, lung interleukin (IL) 6, monocyte chemoattractant protein (MCP)-1 messenger RNA levels, and neutrophil invasion were significantly increased. Liver messenger RNA expression in CLD animals was significantly upregulated for IL-6, tumor necrosis factor alpha, IL-10, and MCP-1 compared with sham and CLP animals. Significantly higher levels of alanine transaminase were observed in CLD animals. Finally, systemic inflammation as measured by plasma IL-6, tumor necrosis factor alpha, IL-1ß, IL-10, and MCP-1 was significantly increased in all CLD animals compared with shams, whereas CLP animals only showed an insignificant increase in the latter molecules. CONCLUSIONS: Our modifications to the classic CLP model significantly produced organ inflammation, liver damage, and a similar mortality compared with a clinical setting, with a reliable onset of sepsis.

Peroxisome proliferator-activated receptor γ-induced T cell apoptosis reduces survival during polymicrobial sepsis.

RATIONALE: Despite intensive research, sepsis displays the most prevalent cause of death on intensive care units. The hallmark of sepsis is an overshooting T-cell death that reduces host defense mechanisms and that is associated with poor patient survival. Previous in vitro studies revealed that the expression of the transcription factor peroxisome proliferator-activated receptor (PPAR) γ was increased in isolated T cells of patients with sepsis. OBJECTIVES: We determined the importance of targeting PPARγ for sepsis treatment and underlying molecular mechanisms for T-cell apoptosis in vivo. METHODS: To mimic human systemic inflammation and septic conditions, we used a nonlethal endotoxemia and a lethal cecum ligation and puncture polymicrobial sepsis model. MEASUREMENTS AND MAIN RESULTS: PPARγ inhibition in T cells with either the PPARγ antagonist GW9662 or a newly generated T cell-specific PPARγ knockout (Tc-PPARγ(-/-)) mice provided a survival advantage during polymicrobial sepsis in mice, which correlated with abrogated T-cell depletion in both in vivo models. Pathway analysis revealed increased antiapoptotic IL-2 and Bcl-2 expression, and activated prosurvival PI3K/Akt signaling under PPARγ-deficient conditions. In line, neutralizing IL-2 in Tc-PPARγ(-/-) mice resulted in T-cell apoptosis and increased mortality. CONCLUSIONS: Our results provide evidence for a pivotal involvement of PPARγ in T-cell depletion by activating two important apoptosis pathways, and subsequently provoking the breakdown of defense mechanisms during systemic inflammation and sepsis.

Hepatocyte growth factor mobilizes non-bone marrow-derived circulating mesoangioblasts.

AIMS: The identification of factors that mobilize subsets of endogenous progenitor cells may provide new therapeutic tools to enhance the repair of ischaemic tissue. We previously identified circulating mesenchymal cells that co-express endothelial markers (so-called circulating mesoangioblasts, cMABs) in children undergoing heart surgery with cardiopulmonary bypass (CPB). However, the mechanisms by which these cells are mobilized and their origin is unclear. METHODS AND RESULTS: Circulating CD73(+)CD45(-)KDR(+) cMABs were analysed in adults undergoing heart surgery with (n = 21) or without CPB (n = 8). During surgery with CPB, cMABs are mobilized with a maximal response at the end of the operation. In contrast, off-pump heart surgery does not stimulate cMAB mobilization, indicating that the stress mediated by CPB induces the mobilization of cMAB. Circulating mesoangioblasts were enriched in blood obtained from the coronary sinus. Histologically, CD73(+) cells were detected around vessels in the heart, indicating that the heart is one of the niches of cMABs. Consistently, studies in gender mismatched bone marrow transplanted patients demonstrated that cMABs did not originate from the bone marrow. Cytokine profiling of serum samples revealed that hepatocyte growth factor (HGF) was profoundly increased at the time point of maximal mobilization of cMABs. Hepatocyte growth factor stimulated the migration of cMABs. Importantly, injection of recombinant HGF increased cMABs in rats. CONCLUSIONS: Hepatocyte growth factor induces mobilization of non-haematopoietic progenitor cells with a cardiac repair capacity. This newly identified function together with the known pleiotrophic effects of HGF makes HGF an attractive therapeutic option for the treatment of ischaemic heart disease.

A review of European guidelines for patient blood management with a particular emphasis on antifibrinolytic drug administration for cardiac surgery.

The concept of patient blood management (PBM) was introduced by the World Health Organization in 2011 and is defined as a "patient-focused, evidence-based and systematic approach for optimizing the management of patients and transfusion of blood products to ensure high quality and effective patient care". Patient blood management is a multimodal approach based on three pillars: optimization of blood mass, minimization of blood loss and optimization of patient tolerance to anaemia. Antifibrinolytics play a major role in cardiac surgery, where the risk of perioperative bleeding is high and affects a majority of patients, by effectively reducing bleeding, transfusions, re-operations, as well as their associated morbidity and mortality. They represent an essential part of the pharmacological arsenal of patient blood management. However, despite the trend towards high-level PBM practices, currently very few European countries have national PBM guidelines and these guidelines, taken as a whole, are heterogeneous in form and content. In particular, the use of antifibrinolytics in cardiac surgery is often not discussed in detail beyond general prophylactic use and any recommendations lack detail including choice of drug, dosing, and mode of administration. Thus, the implementation of PBM programs in Europe is still challenging. In 2021, the WHO published a new document highlighting the urgent need to close the gap in PBM awareness and implementation and announced their upcoming initiative to develop specific PBM implementation guidelines. This review aims first, to summarize the role played by fibrinolysis in haemostatic disorders; second, to give an overview of the current available guidelines in Europe detailing PBM implementation in cardiac surgery; and third, to analyse the place and use of antifibrinolytics in these guidelines.

Thromboprophylaxis with argatroban in critically ill patients with sepsis: a review.

During sepsis, an initial prothrombotic shift takes place, in which coagulatory acute-phase proteins are increased, while anticoagulatory factors and platelet count decrease. Further on, the fibrinolytic system becomes impaired, which contributes to disease severity. At a later stage in sepsis, coagulation factors may become depleted, and sepsis patients may shift into a hypo-coagulable state with an increased bleeding risk. During the pro-coagulatory shift, critically ill patients have an increased thrombosis risk that ranges from developing micro-thromboses that impair organ function to life-threatening thromboembolic events. Here, thrombin plays a key role in coagulation as well as in inflammation. For thromboprophylaxis, low molecular weight heparins (LMWH) and unfractionated heparins (UFHs) are recommended. Nevertheless, there are conditions such as heparin resistance or heparin-induced thrombocytopenia (HIT), wherein heparin becomes ineffective or even puts the patient at an increased prothrombotic risk. In these cases, argatroban, a direct thrombin inhibitor (DTI), might be a potential alternative anticoagulatory strategy. Yet, caution is advised with regard to dosing of argatroban especially in sepsis. Therefore, the starting dose of argatroban is recommended to be low and should be titrated to the targeted anticoagulation level and be closely monitored in the further course of treatment. The authors of this review recommend using DTIs such as argatroban as an alternative anticoagulant in critically ill patients suffering from sepsis or COVID-19 with suspected or confirmed HIT, HIT-like conditions, impaired fibrinolysis, in patients on extracorporeal circuits and patients with heparin resistance, when closely monitored.

Novel aspects of fibrin(ogen) fragments during inflammation.

Coagulation is fundamental for the confinement of infection and/or the inflammatory response to a limited area. Under pathological inflammatory conditions such as arthritis, multiple sclerosis or sepsis, an uncontrolled activation of the coagulation system contributes to inflammation, microvascular failure and organ dysfunction. Coagulation is initiated by the activation of thrombin, which, in turn, triggers fibrin formation by the release of fibrinopeptides. Fibrin is cleaved by plasmin, resulting in clot lysis and an accompanied generation of fibrin fragments such as D and E fragments. Various coagulation factors, including fibrinogen and/or fibrin [fibrin(ogen)] and also fibrin degradation products, modulate the inflammatory response by affecting leukocyte migration and cytokine production. Fibrin fragments are mostly proinflammatory, however, Bß15-42 in particular possesses potential antiinflammatory effects. Bß15-42 inhibits Rho-kinase activation by dissociating Fyn from Rho and, hence prevents stress-induced loss of endothelial barrier function and also leukocyte migration. This article summarizes the state-of-the-art in inflammatory modulation by fibrin(ogen) and fibrin fragments. However, further research is required to gain better understanding of the entire role fibrin fragments play during inflammation and, possibly, disease development.

Biomarkers of endothelial dysfunction: can they help us deciphering systemic inflammation and sepsis?

The endothelial integrity, as mechanical barrier against microorganisms and as natural "anticoagulant", is crucial for physiologic organ function. Systemic activation of the endothelium upon inflammation, sepsis, and septic shock is always ending in blood-tissue barrier disruption. With increasing dysfunction, uncontrolled clotting activation, capillary microthrombi formation, tissue edema, local hypoxia, and ischemia are initiated. This in turn enhances a vicious circle leading to multiple organ failure and death. Therefore, biomarkers reflecting this special compartment may help in the early detection of systemic inflammation and its complications. This review provides an overview of the most important endothelial biomarkers and their possible use in sepsis.

Linking inflammation and coagulation: novel drug targets to treat organ ischemia.

PURPOSE OF REVIEW: Activation of the coagulation system during ischemia/reperfusion injury is an unavoidable event and even further augmented during cardiovascular surgery. Clotting not only leads to disturbance of blood rheology but also enhances the inflammatory response. We aim to highlight the inflammatory properties of the coagulation system and novel potential therapeutic approaches targeting both features. RECENT FINDINGS: Heparin, a thrombin inhibitor, is still the drug of choice for preventing coagulation following, for example, cardiovascular surgery. On the contrary, much effort is done to evaluate the utilization of direct thrombin inhibitors to prevent ischemia/reperfusion injury. Furthermore, targeting the inflammatory potential of the coagulation system seems to be very promising. Fibrin(ogen) and its degradation products modulate the inflammatory response, especially by inducing leukocyte migration. Inhibiting these pro-inflammatory effects, for example, by administration of Bß15-42 was recently shown to be beneficial under various inflammatory conditions. SUMMARY: Ischemia and reperfusion are common activators of coagulation that is also accompanied by inflammation. Therefore, targeting this well orchestrated system might be of therapeutic benefit, as its mode of action is dual: clotting inhibition and anti-inflammation. This novel therapeutic approach might at least be of benefit in the treatment of systemic inflammatory syndromes following, that is, cardiovascular surgery.

A survey of patient blood management for patients undergoing cardiac surgery in nine European countries.

STUDY OBJECTIVE: To describe and compare patient blood management (PBM) practices in cardiac surgery in nine European countries and identify the main risk factors for bleeding or transfusion according to the surveyed centres. DESIGN: We set up an online survey to evaluate PBM practices in two clinical scenarios, risk factors for bleeding or transfusion, and previous experience with antifibrinolytics. SETTING: This survey was completed by European anesthesiologists in 2019. PATIENTS: No patients were included in the survey. INTERVENTION: None. MEASUREMENTS: We evaluated the degree of implementation of PBM practices in patients undergoing cardiac surgery. MAIN RESULTS: Ninety-eight of 177 responses (38%) were complete with variable response rates by country. In a non-emergent situation, no respondents would transfuse red cells preoperatively in an anaemic patient, while cell salvage (89%) and antifibrinolytics (82%) would almost always be used. Optimization of Hemoglobin level (36%) and use of off-pump techniques (34%), minimally invasive surgery (25%) and relatively recently-developed CPB technologies such as mini-bypass (32%) and autologous priming (38%), varied greatly across countries. In an emergent clinical situation, topical haemostatic agents would frequently be used (61%). Tranexamic acid (72%) and aprotinin (20%) were the main antifibrinolytics used, with method of administration and dose varying markedly across countries. Five factors were considered to increase risk of bleeding or transfusion by at least 90% of respondents: pre-operative anaemia, prior cardiac surgery, clopidogrel 5 days or less before surgery, use of other P2Y12 inhibitors at any point, and thrombocytopenia <100.109 platelets/mm3. CONCLUSION: PBM guidelines are not universally implemented in European cardiac surgery centres or countries, resulting in discrepancies in techniques and products used for a given clinical situation.

miR-19a-3p containing exosomes improve function of ischaemic myocardium upon shock wave therapy.

AIMS: As many current approaches for heart regeneration exert unfavourable side effects, the induction of endogenous repair mechanisms in ischaemic heart disease is of particular interest. Recently, exosomes carrying angiogenic miRNAs have been described to improve heart function. However, it remains challenging to stimulate specific release of reparative exosomes in ischaemic myocardium. In the present study, we sought to test the hypothesis that the physical stimulus of shock wave therapy (SWT) causes the release of exosomes. We aimed to substantiate the pro-angiogenic impact of the released factors, to identify the nature of their cargo, and to test their efficacy in vivo supporting regeneration and recovery after myocardial ischaemia. METHODS AND RESULTS: Mechanical stimulation of ischaemic muscle via SWT caused extracellular vesicle (EV) release from endothelial cells both in vitro and in vivo. Characterization of EVs via electron microscopy, nanoparticle tracking analysis and flow cytometry revealed specific exosome morphology and size with the presence of exosome markers CD9, CD81, and CD63. Exosomes exhibited angiogenic properties activating protein kinase b (Akt) and extracellular-signal regulated kinase (ERK) resulting in enhanced endothelial tube formation and proliferation. A miRNA array and transcriptome analysis via next-generation sequencing were performed to specify exosome content. miR-19a-3p was identified as responsible cargo, antimir-19a-3p antagonized angiogenic exosome effects. Exosomes and target miRNA were injected intramyocardially in mice after left anterior descending artery ligation. Exosomes resulted in improved vascularization, decreased myocardial fibrosis, and increased left ventricular ejection fraction as shown by transthoracic echocardiography. CONCLUSION: The mechanical stimulus of SWT causes release of angiogenic exosomes. miR-19a-3p is the vesicular cargo responsible for the observed effects. Released exosomes induce angiogenesis, decrease myocardial fibrosis, and improve left ventricular function after myocardial ischaemia. Exosome release via SWT could develop an innovative approach for the regeneration of ischaemic myocardium.

Colony-stimulating factor-1 transfection of myoblasts improves the repair of failing myocardium following autologous myoblast transplantation.

AIMS: Skeletal myoblasts are used in repair of ischaemic myocardium. However, a large fraction of grafted myoblasts degenerate upon engraftment. Colony-stimulating factor-1 (CSF-1) accelerates myoblast proliferation and angiogenesis. We hypothesized that CSF-1 overexpression improves myoblast survival and cardiac function in ischaemia-induced heart failure. METHODS AND RESULTS: Three weeks following myocardial infarction, rats developed heart failure and received intramyocardial injections of mouse CSF-1-transfected or untransfected primary autologous rat myoblasts, recombinant human CSF-1, mouse CSF-1 expressing plasmids, or culture medium. Tissue gene and protein expression was measured by quantitative RT-PCR (reverse transcription-polymerase chain reaction) and western blotting. Fluorescence imaging and immunocytochemistry were used to analyse myoblasts, endothelial cells, macrophages, and infarct wall thickening. Electrocardiograms were recorded online using a telemetry system. Left ventricular function was assessed by echocardiography over time, and improved significantly only in the CSF-1-overexpressing myoblast group. CSF-1-overexpression enhanced myoblast numbers and was associated with an increased infarct wall thickness, enhanced angiogenesis, increased macrophage recruitment and upregulated matrix metalloproteases (MMP)-2 and -12 in the zone bordering the infarction. Transplantation of CSF-1-overexpressing myoblasts did not result in major arrhythmias. CONCLUSION: Autologous intramyocardial transplantation of CSF-1 overexpressing myoblasts might be a novel strategy in the treatment of ischaemia-induced heart failure.

Colony-stimulating factor-1 antibody reverses chemoresistance in human MCF-7 breast cancer xenografts.

Overexpression of colony-stimulating factor-1 (CSF-1) and its receptor in breast cancer is correlated with poor prognosis. Based on the hypothesis that blockade of CSF-1 would be beneficial in breast cancer treatment, we developed a murinized, polyethylene glycol-linked antigen-binding fragment (Fab) against mouse (host) CSF-1 (anti-CSF-1 Fab). Mice bearing human, chemoresistant MCF-7 breast cancer xenografts were treated with combination chemotherapy (CMF: cyclophosphamide, methotrexate, 5-fluorouracil; cycled twice i.p.), anti-CSF-1 Fab (i.p., cycled every 3 days for 14 days), combined CMF and anti-CSF-1 Fab, or with Ringer's solution as a control. Anti-CSF-1 Fab alone suppressed tissue CSF-1 and retarded tumor growth by 40%. Importantly, in combination with CMF, anti-CSF-1 Fab reversed chemoresistance of MCF-7 xenografts, suppressing tumor development by 56%, down-regulating expression of the chemoresistance genes breast cancer-related protein, multidrug resistance gene 1, and glucosylceramide synthase, and prolonging survival significantly. Combined treatment also reduced angiogenesis and macrophage recruitment and down-regulated tumor matrix metalloproteinase-2 (MMP-2) and MMP-12 expression. These studies support the paradigm of CSF-1 blockade in the treatment of solid tumors and show that anti-CSF-1 antibodies are potential therapeutic agents for the treatment of mammary cancer.

Shock wave treatment after hindlimb ischaemia results in increased perfusion and M2 macrophage presence.

Shock wave therapy (SWT) has been shown to induce angiogenesis in ischaemic muscle. However, the mechanism of action remains unknown. Macrophages are crucial for angiogenic responses after ischaemic injury. The M2 macrophage subset enables tissue repair and induces angiogenesis. It was hypothesized that the angiogenic effects of SWT are at least partly caused by enhanced macrophage recruitment. C57BL/6 mice were subjected to hind limb ischaemia with subsequent SWT or sham treatment. Muscles were analysed via immunofluorescence staining, reverse-transcription polymerase chain reaction and western blot. Gene expression and proteins involved in macrophage recruitment were analysed and tissue sections were stained for macrophages, including subsets, capillaries and arterioles. Laser Doppler perfusion imaging was performed to assess functional outcome. Treated muscles showed increased expression of the pivotal macrophage recruiting factor monocyte chemotactic protein 1 (MCP-1). Higher levels of macrophage marker CD14 were found. Increased numbers of macrophages after SWT could be confirmed by immunofluorescence staining. The expression of the M2 polarization promoting chemokine interleukin 13 was significantly elevated in the treatment group. Elevated mRNA expression of the M2 scavenger receptor CD163 was found after SWT. Immunofluorescence staining confirmed increased numbers of M2 macrophages after treatment. It was found that SWT resulted in higher number of capillaries and arterioles. Assessment of functional outcome revealed significantly improved limb perfusion in treated animals. Shock wave therapy causes increased macrophage recruitment and enhanced polarization towards reparative M2 macrophages in ischaemic muscle resulting in angiogenesis and improved limb perfusion and therefore represents a promising new treatment option for the treatment of ischaemic heart disease. Copyright © 2016 John Wiley & Sons, Ltd.

Valve-sparing root replacement for freestanding pulmonary autograft aneurysm after the Ross procedure.

OBJECTIVE: Autograft dilatation is the main long-term complication following the Ross procedure using the freestanding root replacement technique. We reviewed our 25-year experience with the Ross procedure with a special emphasis on valve-sparing reoperations. METHODS: From 1991 to 2016, 153 patients (29.6 ± 16.6 years; 29.4% pediatric) underwent a Ross operation at our institution with implantation of the autograft as freestanding root replacement. The follow-up is 98.7% complete with a mean of 12.2 ± 5.5 years. RESULTS: Mortality at 30-days was 2.0%. Echocardiography documented no or trivial aortic regurgitation in 99.3% of the patients at discharge. Survival probability at 20 years was 85.4%. No case of autograft endocarditis occurred. Autograft deterioration rate was 2.01% per patient-year, and freedom from autograft reoperation was 75.3% at 15 years. A reoperation for autograft aneurysm was required in 35 patients (22.9%) at a mean interval of 11.1 ± 4.6 years after the Ross procedure. A valve-sparing root replacement was performed in 77% of patients, including 10 David and 17 Yacoub procedures with no early mortality. Three patients required prosthetic valve replacement within 2 years after a Yacoub operation. At latest follow-up, 92% of all surviving patients still carry the pulmonary autograft valve. Freedom from autograft valve replacement was 92.1% at 15 years. CONCLUSIONS: Using the David or Yacoub techniques, the autograft valve can be preserved in the majority of patients with root aneurysms after the Ross procedure. Reoperations can be performed with no early mortality, a good functional midterm result, and an acceptable reintervention rate.

Shock Wave Therapy Improves Cardiac Function in a Model of Chronic Ischemic Heart Failure: Evidence for a Mechanism Involving VEGF Signaling and the Extracellular Matrix.

Background Mechanical stimulation of acute ischemic myocardium by shock wave therapy ( SWT ) is known to improve cardiac function by induction of angiogenesis. However, SWT in chronic heart failure is poorly understood. We aimed to study whether mechanical stimulation upon SWT improves heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis and to dissect underlying mechanisms. Methods and Results SWT was applied in a mouse model of chronic myocardial ischemia. To study effects of SWT on postnatal vasculogenesis, wild-type mice received bone marrow transplantation from green fluorescence protein donor mice. Underlying mechanisms were elucidated in vitro in endothelial cells and murine aortic rings. Echocardiography and pressure/volume measurements revealed improved left ventricular ejection fraction, myocardial contractility, and diastolic function and decreased myocardial fibrosis after treatment. Concomitantly, numbers of capillaries and arterioles were increased. SWT resulted in enhanced expression of the chemoattractant stromal cell-derived factor 1 in ischemic myocardium and serum. Treatment induced recruitment of bone marrow-derived endothelial cells to the site of injury. In vitro, SWT resulted in endothelial cell proliferation, enhanced survival, and capillary sprouting. The effects were vascular endothelial growth factor receptor 2 and heparan sulfate proteoglycan dependent. Conclusions SWT positively affects heart function in chronic ischemic heart failure by induction of angiogenesis and postnatal vasculogenesis. SWT upregulated pivotal angiogenic and vasculogenic factors in the myocardium in vivo and induced proliferative and anti-apoptotic effects on endothelial cells in vitro. Mechanistically, these effects depend on vascular endothelial growth factor signaling and heparan sulfate proteoglycans. SWT is a promising treatment option for regeneration of ischemic myocardium.