Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described. Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin. Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Interleukin-1β (IL-1β) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 β are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1β. Out of the 4 major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human heart failure regardless of the etiology (ischemic or dilated cardiomyopathy) while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human THP-1 monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in heart failure and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning of probenecid for HF indications.

Background and Purpose: Immune checkpoint inhibitors (ICI), such as anti‐PD‐1 monoclonal antibodies, have revolutionized cancer therapy by enhancing the cytotoxic effects of T‐cells against tumours. However, enhanced T‐cell activity also may cause myocarditis and cardiotoxicity. Our understanding of the mechanisms of ICI‐induced cardiotoxicity is limited. Here, we aimed to investigate the effect of PD‐1 inhibition on cardiac function and explore the molecular mechanisms of ICI‐induced cardiotoxicity.Experimental Approach: C57BL6/J and BALB/c mice were treated with isotype control or anti‐PD‐1 antibody.Echocardiography was used to assess cardiac function. Cardiac transcriptomic changes were investigated by bulk RNA sequencing. Inflammatory changes were assessed by qRT‐PCR and immunohistochemistry in heart, thymus, and spleen of the animals. In follow‐up experiments, anti‐CD4 and anti‐IL‐17A antibodies were used along with PD‐1 blockade in C57BL/6J mice.Key Results: Anti‐PD‐1 treatment led to cardiac dysfunction and left ventricular dilation in C57BL/6J mice, with increased nitrosative stress. Only mild inflammation was observed in the heart. However, PD‐1 inhibition resulted in enhanced thymic inflammatory signalling, whereIl17aincreased most prominently. In BALB/c mice, cardiac dysfunction was not evident, and thymic inflammatory activation was more balanced. Inhibition of IL‐17A prevented anti‐PD‐1‐induced cardiac dysfunction in C57BL6/J mice. Comparing myocardial transcriptomic changes in C57BL/6J and BALB/c mice, differentially regulated genes (Dmd,Ass1,Chrm2,Nfkbia,Stat3,Gsk3b,Cxcl9,Fxyd2, andLdb3) were revealed, related to cardiac structure, signalling, and inflammation.Conclusions: PD‐1 blockade induces cardiac dysfunction in mice with increased IL‐17 signalling in the thymus. Pharmacological inhibition of IL‐17A treatment prevents ICI‐induced cardiac dysfunction.

Remote ischemic perconditioning (RIPerC) has a promising therapeutic insight to improve the prognosis of acute myocardial infarction. Chronic comorbidities such as diabetes are known to interfere with conditioning interventions by modulating cardioprotective signaling pathways, such as e.g., mTOR pathway and autophagy. However, the effect of acute hyperglycemia on RIPerC has not been studied so far. Therefore, here we investigated the effect of acute hyperglycemia on cardioprotection by RIPerC. Wistar rats were divided into normoglycemic (NG) and acute hyperglycemic (AHG) groups. Acute hyperglycemia was induced by glucose infusion to maintain a serum glucose concentration of 15–20 mM throughout the experimental protocol. NG rats received mannitol infusion of an equal osmolarity. Both groups were subdivided into an ischemic (Isch) and a RIPerC group. Each group underwent reversible occlusion of the left anterior descending coronary artery (LAD) for 40 min in the presence or absence of acute hyperglycemia. After the 10-min LAD occlusion, RIPerC was induced by 3 cycles of 5-min unilateral femoral artery and vein occlusion and 5-min reperfusion. After 120 min of reperfusion, infarct size was measured by triphenyltetrazolium chloride staining. To study underlying signaling mechanisms, hearts were harvested for immunoblotting after 35 min in both the NG and AHG groups. Infarct size was significantly reduced by RIPerC in NG, but not in the AHG group (NG + Isch: 46.27 ± 5.31 % vs. NG + RIPerC: 24.65 ± 7.45 %, p < 0.05; AHG + Isch: 54.19 ± 4.07 % vs. 52.76 ± 3.80 %). Acute hyperglycemia per se did not influence infarct size, but significantly increased the incidence and duration of arrhythmias. Acute hyperglycemia activated mechanistic target of rapamycine (mTOR) pathway, as it significantly increased the phosphorylation of mTOR and S6 proteins and the phosphorylation of AKT. In spite of a decreased LC3II/LC3I ratio, other markers of autophagy, such as ATG7, ULK1 phopsphorylation, Beclin 1 and SQSTM1/p62, were not modulated by acute hyperglycemia. Furthermore, acute hyperglycemia significantly elevated nitrative stress in the heart (0.87 ± 0.01 vs. 0.50 ± 0.04 µg 3-nitrotyrosine/mg protein, p < 0.05). This is the first demonstration that acute hypreglycemia deteriorates cardioprotection by RIPerC. The mechanism of this phenomenon may involve an acute hyperglycemia-induced increase in nitrative stress and activation of the mTOR pathway.

The available pharmacological options in the management of cardiovascular diseases such as ischaemic heart disease and subsequent heart failure are effective in slowing the progression of this condition. However, the long‐term prognosis is still poor, raising the demand for new therapeutic strategies. Drug repurposing is a time‐ and cost‐effective drug development strategy that offers approved and abandoned drugs a new chance for new indications. Recently, drugs used for the management of gout‐related inflammation such as canakinumab or colchicine have been considered for drug repurposing in cardiovascular indications. The old uricosuric drug, probenecid, has been identified as a novel therapeutic option in the management of specific cardiac diseases as well. Probenecid can modulate myocardial contractility and vascular tone and exerts anti‐inflammatory properties. The mechanisms behind these beneficial effects might be related inhibition of inflammasomes, and to modulation purinergic–pannexin‐1 signalling and TRPV2 channels, which are recently identified molecular targets of probenecid. In this review, we provide an overview on repurposing probenecid for ischaemic heart disease and subsequent heart failure by summarizing the related experimental and clinical data and propose its potential repurposing to treat cardiovascular diseases.