Acute hyperglycemia abolishes cardioprotection by remote ischemic perconditioning

BACKGROUND: 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. METHODS: 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. RESULTS: 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 microg 3-nitrotyrosine/mg protein, p < 0.05). CONCLUSIONS: 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

Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods

BACKGROUND: 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. AIM: Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC). METHODS AND RESULTS: 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 degrees 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 degrees C, or UC performed at 37 degrees 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. CONCLUSION: Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield

Hypercholesterolemia downregulates autophagy in the rat heart

Background: We have previously shown that efficiency of ischemic conditioning is diminished in hypercholesterolemia and that autophagy is necessary for cardioprotection. However, it is unknown whether isolated hypercholesterolemia disturbs autophagy or the mammalian target of rapamycin (mTOR) pathways. Therefore, we investigated whether isolated hypercholesterolemia modulates cardiac autophagy-related pathways or programmed cell death mechanisms such as apoptosis and necroptosis in rat heart. Methods: Male Wistar rats were fed either normal chow (NORM; n=9) or with 2% cholesterol and 0.25% cholic acid-enriched diet (CHOL; n=9) for 12 weeks. CHOL rats exhibited a 41% increase in plasma total cholesterol level over that of NORM rats (4.09mmol/L vs. 2.89mmol/L) at the end of diet period. Animals were sacrificed, hearts were excised and briefly washed out. Left ventricles were snap-frozen for determination of markers of autophagy, mTOR pathway, apoptosis, and necroptosis by Western blot. Results: Isolated hypercholesterolemia was associated with a significant reduction in expression of cardiac autophagy markers such as LC3-II, Beclin-1, Rubicon and RAB7 as compared to controls. Phosphorylation of ribosomal S6, a surrogate marker for mTOR activity, was increased in CHOL samples. Cleaved caspase-3, a marker of apoptosis, increased in CHOL hearts, while no difference in the expression of necroptotic marker RIP1, RIP3 and MLKL was detected between treatments. Conclusions: This is the first comprehensive analysis of autophagy and programmed cell death pathways of apoptosis and necroptosis in hearts of hypercholesterolemic rats. Our data show that isolated hypercholesterolemia suppresses basal cardiac autophagy and that the decrease in autophagy may be a result of an activated mTOR pathway. Reduced autophagy was accompanied by increased apoptosis, while cardiac necroptosis was not modulated by isolated hypercholesterolemia. Decreased basal autophagy and elevated apoptosis may be responsible for the loss of cardioprotection reported in hypercholesterolemic animals

MicroRNA expression as risk biomarker of breast cancer metastasis : a pilot retrospective case-cohort study

Background: MicroRNAs (miRNAs) are small, non-coding RNA molecules involved in post-transcriptional gene regulation and have recently been shown to play a role in cancer metastasis. In solid tumors, especially breast cancer, alterations in miRNA expression contribute to cancer pathogenesis, including metastasis. Considering the emerging role of miRNAs in metastasis, the identification of predictive markers is necessary to further the understanding of stage-specific breast cancer development. This is a retrospective analysis that aimed to identify molecular biomarkers related to distant breast cancer metastasis development. Methods: A retrospective case cohort study was performed in 64 breast cancer patients treated during the period from 1998-2001. The case group (n = 29) consisted of patients with a poor prognosis who presented with breast cancer recurrence or metastasis during follow up. The control group (n = 35) consisted of patients with a good prognosis who did not develop breast cancer recurrence or metastasis. These patient groups were stratified according to TNM clinical stage (CS) I, II and III, and the main clinical features of the patients were homogeneous. MicroRNA profiling was performed and biomarkers related to metastatic were identified independent of clinical stage. Finally, a hazard risk analysis of these biomarkers was performed to evaluate their relation to metastatic potential. Results: MiRNA expression profiling identified several miRNAs that were both specific and shared across all clinical stages (p <= 0.05). Among these, we identified miRNAs previously associated with cell motility (let-7 family) and distant metastasis (hsa-miR-21). In addition, hsa-miR-494 and hsa-miR-21 were deregulated in metastatic cases of CSI and CSII. Furthermore, metastatic miRNAs shared across all clinical stages did not present high sensitivity and specificity when compared to specific-CS miRNAs. Between them, hsa-miR-183 was the most significative of CSII, which miRNAs combination for CSII (hsa-miR-494, hsa-miR-183 and hsa-miR-21) was significant and were a more effective risk marker compared to the single miRNAs. Conclusions: Women with metastatic breast cancer, especially CSII, presented up-regulated levels of miR-183, miR-494 and miR-21, which were associated with a poor prognosis. These miRNAs therefore represent new risk biomarkers of breast cancer metastasis and may be useful for future targeted therapies.We thank the Researcher Support Center of Barretos Cancer Hospital, especially the statistician Zanardo C. for assisting in the statistical analysis.This study received financial support from Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (Fapesp, Proc: 10/ 16796-0, Sao Paulo, Brazil)

Remote ischemic conditioning: from experimental observation to clinical application: report from the 8th Biennial Hatter Cardiovascular Institute Workshop

In 1993, Przyklenk and colleagues made the intriguing experimental observation that 'brief ischemia in one vascular bed also protects remote, virgin myocardium from subsequent sustained coronary artery occlusion' and that this effect '.... may be mediated by factor(s) activated, produced, or transported throughout the heart during brief ischemia/reperfusion'. This seminal study laid the foundation for the discovery of 'remote ischemic conditioning' (RIC), a phenomenon in which the heart is protected from the detrimental effects of acute ischemia/reperfusion injury (IRI), by applying cycles of brief ischemia and reperfusion to an organ or tissue remote from the heart. The concept of RIC quickly evolved to extend beyond the heart, encompassing inter-organ protection against acute IRI. The crucial discovery that the protective RIC stimulus could be applied non-invasively, by simply inflating and deflating a blood pressure cuff placed on the upper arm to induce cycles of brief ischemia and reperfusion, has facilitated the translation of RIC into the clinical setting. Despite intensive investigation over the last 20 years, the underlying mechanisms continue to elude researchers. In the 8th Biennial Hatter Cardiovascular Institute Workshop, recent developments in the field of RIC were discussed with a focus on new insights into the underlying mechanisms, the diversity of non-cardiac protection, new clinical applications, and large outcome studies. The scientific advances made in this field of research highlight the journey that RIC has made from being an intriguing experimental observation to a clinical application with patient benefit

Possible involvement of caveolin in attenuation of cardioprotective effect of ischemic preconditioning in diabetic rat heart

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) has been noted to produce ischemic preconditioning (IPC)-mediated cardioprotection. Caveolin is a negative regulator of NO, which inhibits endothelial nitric oxide synthase (eNOS) by making caveolin-eNOS complex. The expression of caveolin is increased during diabetes mellitus (DM). The present study was designed to investigate the involvement of caveolin in attenuation of the cardioprotective effect of IPC during DM in rat.</p> <p>Methods</p> <p>Experimental DM was induced by single dose of streptozotocin (50 mg/Kg, <it>i.p</it>,) and animals were used for experiments four weeks later. Isolated heart was mounted on Langendorff's apparatus, and was subjected to 30 min of global ischemia and 120 min of reperfusion. IPC was given by four cycles of 5 min of ischemia and 5 min of reperfusion with Kreb's-Henseleit solution (K-H). Extent of injury was measured in terms of infarct size by triphenyltetrazolium chloride (TTC) staining, and release of lactate dehydrogenase (LDH) and creatin kinase-MB (CK-MB) in coronary effluent. The cardiac release of NO was noted by measuring the level of nitrite in coronary effluent.</p> <p>Results</p> <p>IPC- induced cardioprotection and release of NO was significantly decreased in diabetic rat heart. Pre-treatment of diabetic rat with daidzein (DDZ) a caveolin inhibitor (0.2 mg/Kg/s.c), for one week, significantly increased the release of NO and restored the attenuated cardioprotective effect of IPC. Also perfusion of sodium nitrite (10 μM/L), a precursor of NO, significantly restored the lost effect of IPC, similar to daidzein in diabetic rat. Administration of 5-hydroxy deaconate (5-HD), a mito K_ATPchannel blocker, significantly abolished the observed IPC-induced cardioprotection in normal rat or daidzein and sodium nitrite perfused diabetic rat heart alone or in combination.</p> <p>Conclusions</p> <p>Thus, it is suggested that attenuation of the cardioprotection in diabetic heart may be due to decrease the IPC mediated release of NO in the diabetic myocardium, which may be due to up -regulation of caveolin and subsequently decreased activity of eNOS.</p

Corrigendum to "European contribution to the study of ROS:A summary of the findings and prospects for the future from the COST action BM1203 (EU-ROS)" [Redox Biol. 13 (2017) 94-162]

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment