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Inflammasomes are crucial gatekeepers of the immune response, but their maladaptive activation associates with inflammatory pathologies. Besides canonical activation, monocytes can trigger non-transcriptional or rapid inflammasome activation that has not been well defined in the context of acute myocardial infarction (AMI). Rapid transcription-independent inflammasome activation induced by simultaneous TLR priming and triggering stimulus was measured by caspase-1 (CASP1) activity and interleukin release. Both classical and intermediate monocytes from healthy donors exhibited robust CASP1 activation, but only classical monocytes produced high mature interleukin-18 (IL18) release. We also recruited a limited number of coronary artery disease (CAD, n=31) and AMI (n=29) patients to evaluate their inflammasome function and expression profiles. Surprisingly, monocyte subpopulations isolated from blood collected during percutaneous coronary intervention (PCI) from AMI patients presented diminished CASP1 activity and abrogated IL18 release despite increased NLRP3 gene expression. This unexpected attenuated rapid inflammasome activation was accompanied by a significant increase of TNFAIP3 and IRAKM expression. Moreover, TNFAIP3 protein levels of circulating monocytes showed positive correlation with high sensitive troponin T (hsTnT), implying an association between TNFAIP3 upregulation and the severity of tissue injury. We suggest this monocyte attenuation to be a protective phenotype aftermath following a very early inflammatory wave in the ischemic area. Damage-associated molecular patterns (DAMPs) or other signals trigger a transitory negative feedback loop within newly recruited circulating monocytes as a mechanism to reduce post-injury tissue damage.

BackgroundHemostasis and inflammation are both dysregulated in patients with moderate-to-severe coronavirus disease 2019 (COVID-19). Yet, both processes can also be disturbed in patients with other respiratory diseases, and the interactions between coagulation, inflammation, and disease severity specific to COVID-19 are still vague.MethodsHospitalized patients with acute respiratory symptoms and with severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)-positive (COVpos) and SARS-CoV2-negative (COVneg) status were included. We assessed adenosine diphosphate (ADP)-, thrombin receptor activator peptide 6 (TRAP)-, and arachidonic acid (AA)-induced platelet reactivity by impedance aggregometry, as well as leukocyte subtype spectrum and platelet-leukocyte aggregates by flow cytometry and inflammatory cytokines by cytometric bead array.ResultsADP-, TRAP-, and AA-induced platelet reactivity was significantly higher in COVpos than in COVneg patients. Disease severity, assessed by sequential organ failure assessment (SOFA) score, was higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. The SOFA score correlated significantly with the mean platelet volume and TRAP-induced platelet aggregability. A larger percentage of classical and intermediate monocytes, and of CD4pos T cells (TH) aggregated with platelets in COVpos than in COVneg patients. Interleukin (IL)-1 receptor antagonist (RA) and IL-6 levels were higher in COVpos than in COVneg patients and again higher in deceased COVpos patients than in surviving COVpos. IL-1RA and IL-6 levels correlated with the SOFA score in COVpos but not in COVneg patients. In both respiratory disease groups, absolute levels of B-cell-platelet aggregates and NK-cell-platelet aggregates were correlated with ex vivo platelet aggegation upon stimulation with AA and ADP, respectively, indicating a universal, but not a COVID-19-specific mechanism.ConclusionIn moderate-to-severe COVID-19, but not in other respiratory diseases, disease severity was associated with platelet hyperreactivity and a typical inflammatory signature. In addition to a severe inflammatory response, platelet hyperreactivity associated to a worse clinical outcome in patients with COVID-19, pointing to the importance of antithrombotic therapy for reducing disease severity.

Objective: Extracellular vesicles (EV) are sub-1 μm bilayer lipid coated particles and have been shown play a role in long-term cardiovascular outcome after ischemic stroke. However, the dynamic change of EV after stroke and their implications for functional outcome have not yet been elucidated.Methods: Serial blood samples from 110 subacute ischemic stroke patients enrolled in the prospective BAPTISe study were analyzed. All patients participated in the PHYS-STROKE trial and received 4-week aerobic training or relaxation sessions. Levels of endothelial-derived (EnV: Annexin V+, CD45–, CD41–, CD31+/CD144+/CD146+), leukocyte-derived (LV: Annexin V+, CD45+, CD41–), monocytic-derived (MoV: Annexin V+, CD41–, CD14+), neuronal-derived (NV: Annexin V+, CD41–, CD45–, CD31–, CD144–, CD146–, CD56+/CD171+/CD271+), and platelet-derived (PV: Annexin V+, CD41+) EV were assessed via fluorescence-activated cell sorting before and after the trial intervention. The levels of EV at baseline were dichotomized at the 75th percentile, with the EV levels at baseline above the 75th percentile classified as “high” otherwise as “low.” The dynamic of EV was classified based on the difference between baseline and post intervention, defining increases above the 75th percentile as “high increase” otherwise as “low increase.” Associations of baseline levels and change in EV concentrations with Barthel Index (BI) and cardiovascular events in the first 6 months post-stroke were analyzed using mixed model regression analyses and cox regression.Results: Both before and after intervention PV formed the largest population of vesicles followed by NV and EnV. In mixed-model regression analyses, low NV [−8.57 (95% CI −15.53 to −1.57)] and low PV [−6.97 (95% CI −13.92 to −0.01)] at baseline were associated with lower BI in the first 6 months post-stroke. Patients with low increase in NV [8.69 (95% CI 2.08–15.34)] and LV [6.82 (95% CI 0.25–13.4)] were associated with reduced BI in the first 6 months post-stroke. Neither baseline vesicles nor their dynamic were associated with recurrent cardiovascular events.Conclusion: This is the first report analyzing the concentration and the dynamic of EV regarding associations with functional outcome in patients with subacute stroke. Lower levels of PV and NV at baseline were associated with a worse functional outcome in the first 6 months post-stroke. Furthermore, an increase in NV and LV over time was associated with worse BI in the first 6 months post-stroke. Further investigation of the relationship between EV and their dynamic with functional outcome post-stroke are warranted.Clinical Trial Registration:clinicaltrials.gov/, identifier: NCT01954797.

Background: Protease-activated receptor 1 (PAR1) and toll-like receptors (TLRs) are inflammatory mediators contributing to atherogenesis and atherothrombosis. Vorapaxar, which selectively antagonizes PAR1-signaling, is an approved, add-on antiplatelet therapy for secondary prevention. The non-hemostatic, platelet-independent, pleiotropic effects of vorapaxar have not yet been studied. Methods and Results: Cellular targets of PAR1 signaling in the vasculature were identified in three patient cohorts with atherosclerotic disease. Evaluation of plasma biomarkers (n = 190) and gene expression in endomyocardial biopsies (EMBs) (n = 12) revealed that PAR1 expression correlated with endothelial activation and vascular inflammation. PAR1 colocalized with TLR2/4 in human carotid plaques and was associated with TLR2/4 gene transcription in EMBs. In addition, vorapaxar reduced atherosclerotic lesion size in apolipoprotein E–knock out (ApoEko) mice. This reduction was associated with reduced expression of vascular adhesion molecules and TLR2/4 presence, both in isolated murine endothelial cells and the aorta. Thrombin-induced uptake of oxLDL was augmented by additional TLR2/4 stimulation and abrogated by vorapaxar. Plaque-infiltrating pro-inflammatory cells were reduced in vorapaxar-treated ApoEko mice. A shift toward M2 macrophages paralleled a decreased transcription of pro-inflammatory cytokines and chemokines. Conclusions: PAR1 inhibition with vorapaxar may be effective in reducing residual thrombo-inflammatory event risk in patients with atherosclerosis independent of its effect on platelets.

Adenine nucleotide translocase 1 (ANT1) transfers ATP and ADP over the mitochondrial inner membrane and thus supplies the cell with energy. This study analyzed the role of ANT1 in the immune response of ischemic heart tissue. Ischemic ANT1 overexpressing hearts experienced a shift toward an anti-inflammatory immune response. The shift was characterized by low interleukin (IL)-1β expression and M1 macrophage infiltration, whereas M2 macrophage infiltration and levels of IL-10, IL-4, and transforming growth factor (TGFβ) were increased. The modulated immune response correlated with high mitochondrial integrity, reduced oxidative stress, low left ventricular end-diastolic heart pressure, and a high survival rate. Isolated ANT1-transgenic (ANT1-TG) cardiomyocytes expressed low levels of pro-inflammatory cytokines such as IL-1α, tumor necrosis factor α, and TGFβ. However, they showed increased expression and cellular release of anti-inflammatory immunomodulators such as vascular endothelial growth factor. The secretome from ANT1-TG cardiomyocytes initiated stress resistance when applied to ischemic wild-type cardiomyocytes and endothelial cells. It additionally prevented macrophages from expressing pro-inflammatory cytokines. Additionally, ANT1 expression correlated with genes that are related to cytokine and growth factor pathways in hearts of patients with ischemic cardiomyopathy. In conclusion, ANT1-TG cardiomyocytes secrete soluble factors that influence ischemic cardiac cells and initiate an anti-inflammatory immune response in ischemic hearts.

Induced pluripotent stem cells (iPSCs) avoid many of the restrictions that hamper the application of human embryonic stem cells: limited availability of source material due to legal restrictions in some countries, immunogenic rejection and ethical concerns. Also, the donor’s clinical phenotype is often known when working with iPSCs. Therefore, iPSCs seem ideal to tackle the two biggest tasks of regenerative medicine: degenerative diseases with genetic cause (e.g., Duchenne’s muscular dystrophy) and organ replacement in age-related diseases (e.g., end-stage heart or renal failure), especially in combination with recently developed gene-editing tools. In the setting of autologous transplantation in elderly patients, donor age becomes a potentially relevant factor that needs to be assessed. Here, we review and critically discuss available data pertinent to the questions: How does donor age influence the reprogramming process and iPSC functionality? Would it even be possible to reprogram senescent somatic cells? How does donor age affect iPSC differentiation into specialised cells and their functionality? We also identify research needs, which might help resolve current unknowns. Until recently, most hallmarks of ageing were attributed to an accumulation of DNA damage over time, and it was thus expected that DNA damage from a somatic cell would accumulate in iPSCs and the cells derived from them. In line with this, a decreased lifespan of cloned organisms compared with the donor was also observed in early cloning experiments. Therefore, it was questioned for a time whether iPSC derived from an old individual’s somatic cells would suffer from early senescence and, thus, may not be a viable option either for disease modelling nor future clinical applications. Instead, typical signs of cellular ageing are reverted in the process of iPSC reprogramming, and iPSCs from older donors do not show diminished differentiation potential nor do iPSC-derived cells from older donors suffer early senescence or show functional impairments when compared with those from younger donors. Thus, the data would suggest that donor age does not limit iPSC application for modelling genetic diseases nor regenerative therapies. However, open questions remain, e.g., regarding the potential tumourigenicity of iPSC-derived cells and the impact of epigenetic pattern retention.

Introduction. DPP4 inhibitors (gliptins) are commonly used antidiabetic drugs for the treatment of type 2 diabetes. Gliptins also act in a glucose-independent manner and show vasoregenerative effects. We have shown that gliptins can remarkably accelerate vascular healing after vascular injury. However, the underlying mechanisms remain unclear. Here, we examined potential signaling pathways linking gliptins to enhanced endothelial regeneration. Methods and Results. We used wild-type and GLP1 receptor knockout (Glp1r−/−) mice to investigate the underlying mechanisms of gliptin-induced reendothelialization. The prototype DPP4 inhibitor sitagliptin accelerated endothelial healing in both animal models. Improved endothelial growth was associated with gliptin-mediated progenitor cell recruitment into the diseased vascular wall via the SDF1-CXCR4 axis independent of GLP1R-dependent signaling pathways. Furthermore, SDF1 showed direct proproliferative effects on endothelial cells. Excessive neointimal formation was not observed in gliptin- or placebo-treated Glp1r−/− mice. Conclusion. We identified the SDF1-CXCR4 axis as a crucial signaling pathway for endothelial regeneration after acute vascular injury. Furthermore, SDF1 can directly increase endothelial cell proliferation. Gliptin-mediated potentiation of endothelial regeneration was preserved in Glp1r−/− animals. Thus, gliptin-mediated endothelial regeneration proceeds through SDF-1/CXCR4 in a GLP1R-independent manner after acute vascular injury.

Little is known either about either physical activity patterns, or other lifestyle-related prevention measures in heart transplantation (HTx) recipients. The history of HTx started more than 50 years ago but there are still no guidelines or position papers highlighting the features of prevention and rehabilitation after HTx. The aims of this scientific statement are (i) to explain the importance of prevention and rehabilitation after HTx, and (ii) to promote the factors (modifiable/non-modifiable) that should be addressed after HTx to improve patients’ physical capacity, quality of life and survival. All HTx team members have their role to play in the care of these patients and multidisciplinary prevention and rehabilitation programmes designed for transplant recipients. HTx recipients are clearly not healthy disease-free subjects yet they also significantly differ from heart failure patients or those who are supported with mechanical circulatory support. Therefore, prevention and rehabilitation after HTx both need to be specifically tailored to this patient population and be multidisciplinary in nature. Prevention and rehabilitation programmes should be initiated early after HTx and continued during the entire post-transplant journey. This clinical consensus statement focuses on the importance and the characteristics of prevention and rehabilitation designed for HTx recipients.

Atherosclerosis is an inflammatory disease characterized by accumulation of leukocytes in the arterial intima. Members of the selectin family of adhesion molecules are important mediators of leukocyte extravasation. However, it is unclear whether L-selectin (L-sel) is involved in the pathogenesis of atherosclerosis. In the present study, mice deficient in L-selectin (L-sel(-/-)) animals were crossed with mice lacking Apolipoprotein E (ApoE(-/-)). The development of atherosclerosis was analyzed in double-knockout ApoE/L-sel (ApoE(-/-)L-sel(-/-)) mice and the corresponding ApoE(-/-) controls fed either a normal or a high cholesterol diet (HCD). After 6 weeks of HCD, aortic lesions were increased two-fold in ApoE(-/-)L-sel(-/-) mice as compared to ApoE(-/-) controls (2.46%±0.54% vs 1.28%±0.24% of total aortic area; p

We aimed to understand early alterations in kinin-mediated migration of circulating angio-supportive cells and dysfunction of kinin-sensitive cells in type-1 diabetic (T1D) patients before the onset of cardiovascular disease.Total mononuclear cells (MNC) were isolated from peripheral blood of 28 T1D patients free from cardiovascular complications except mild background retinopathy (age: 34.8+/-1.6 years, HbA(1C): 7.9+/-0.2%) and 28 age- and sex-matched non-diabetic controls (H). We tested expression of kinin receptors by flow cytometry and migratory capacity of circulating monocytes and progenitor cells towards bradykinin (BK) in transwell migration assays. MNC migrating towards BK (BK(mig)) were assessed for capacity to support endothelial cell function in a matrigel assay, as well as generation of nitric oxide (NO) and superoxide (O(2) (-)*) by using the fluorescent probes diaminofluorescein and dihydroethidium.CD14(hi)CD16(neg), CD14(hi)CD16(pos) and CD14(lo)CD16(pos) monocytes and circulating CD34(pos) progenitor cells did not differ between T1D and H subjects in their kinin receptor expression and migration towards BK. T1D BK(mig) failed to generate NO upon BK stimulation and supported endothelial cell network formation less efficiently than H BK(mig). In contrast, O(2) (-)* production was similar between groups. High glucose disturbed BK-induced NO generation by MNC-derived cultured angiogenic cells.Our data point out alterations in kinin-mediated functions of circulating MNC from T1D patients, occurring before manifest macrovascular damage or progressed microvascular disease. Functional defects of MNC recruited to the vessel wall might compromise endothelial maintenance, initially without actively promoting endothelial damage, but rather by lacking supportive contribution to endothelial regeneration and healing.

Abstract “Medical scientists” are postgraduate investigators who are engaged in biomedical research, and either hold a biomedical PhD or are qualified in medicine but do not participate in patient care. Medical scientists constitute ~40% of staff at medical faculties and >90% at nonuniversity medical research institutions in Germany. However, medical scientists in Germany face limited long‐term career prospects and a lack of dedicated training and support programmes. They also face time limits on their career progression arising from national academic employment legislation, and imminent reforms by the German government are likely to make this worse. Nevertheless, recent developments in the educational landscape including the introduction of increasingly focused MSc, pre‐PhD, and doctoral programmes to train medically aware basic scientists, as well as improved general recognition of the roles and relevance of medical scientists in health research, are encouraging. Physiologists have taken essential steps to improve the recognition of medical scientists in Germany by introducing a “specialist physiologist” qualification; this initiative could be applied to support medical scientists in other fields and countries. In this review, we describe the particular challenges facing medical scientists in Germany and make recommendations that may apply to other academic systems.

BackgroundMetabolic syndrome (MetS) is defined by the presence of central obesity plus ≥two metabolic/cardiovascular risk factors (RF), with inflammation being a major disease-driving mechanism. Structured endurance exercise training (ET) may positively affect these traits, as well as cardiorespiratory fitness (V̇O2peak).AimsWe explore individual ET-mediated improvements of MetS-associated RF in relation to improvements in V̇O2peak and inflammatory profile.MethodsMetS patients from two randomized controlled trials, ExMET (n = 24) and OptimEx (n = 34), had performed 4- or 3-months supervised ET programs according to the respective trial protocol. V̇O2peak, MetS-defining RFs (both RCTs), broad blood leukocyte profile, cytokines and plasma proteins (ExMET only) were assessed at baseline and follow-up. Intra-individual changes in RFs were analysed for both trials separately using non-parametric approaches. Associations between changes in each RF over the exercise period (n-fold of baseline values) were correlated using a non-parametrical approach (Spearman). RF clustering was explored by uniform manifold approximation and projection (UMAP) and changes in RF depending on other RF or exercise parameters were explored by recursive partitioning.ResultsFour months of ET reduced circulating leukocyte counts (63.5% of baseline, P = 8.0e-6), especially effector subtypes. ET response of MetS-associated RFs differed depending on patients’ individual RF constellation, but was not associated with individual change in V̇O2peak. Blood pressure lowering depended on cumulative exercise duration (ExMET: ≥102 min per week; OptimEx-MetS: ≥38 min per session) and baseline triglyceride levels (ExMET:

Inflammation-induced coagulopathy is a common complication associated with coronavirus disease 2019 (COVID-19). We aim to evaluate the association of NETosis and complement markers with each other as well as their association with thrombogenicity and disease severity in COVID-19. The study included hospitalized patients with an acute respiratory infection: patients with SARS-CoV2 infection (COVpos, n = 47) or either pneumonia or infection-triggered acute exacerbated COPD (COVneg, n = 36). Our results show that NETosis, coagulation, and platelets, as well as complement markers, were significantly increased in COVpos patients, especially in severely ill COVpos patients. NETosis marker MPO/DNA complexes correlated with coagulation, platelet, and complement markers only in COVpos. Severely ill COVpos patients showed an association between complement C3 and SOFA (R = 0.48; p ≤ 0.028), C5 and SOFA (R = 0.46; p ≤ 0.038), and C5b-9 and SOFA (R = 0.44; p ≤ 0.046). This study provides further evidence that NETosis and the complement system are key players in COVID-19 inflammation and clinical severity. Unlike previous studies that found NETosis and complement markers to be elevated in COVID-19 patients compared to healthy controls, our findings show that this characteristic distinguishes COVID-19 from other pulmonary infectious diseases. Based on our results, we propose that COVID-19 patients at high risk for immunothrombosis could be identified via elevated complement markers such as C5.

Abstract See related articles in EMBO Molecular Medicine http://dx.doi.org/10.1002/emmm.201302695 and http://dx.doi.org/10.1002/emmm.201302752