A szívizom sztressz-adaptációja: a peroxinitrit, a mátrix metalloproteinázok, és a hiperlipidémia szerepe = Stress adaptation of the myocardium: role of peroxynitrite, matrix metalloproteinases, and hyperlipidemia

A hiperlipidémia talaján kialakuló iszkémiás szívbetegség a leggyakoribb halálokok közé tartozik. A 4 éves project során a szívizom iszkémiának és az iszkémiás stressz adaptációs képességének (iszkémiás pre- és posztkondíció) celluláris mechanizmusait vizsgáltuk állatkísérletekben, különösképpen a peroxinitrit és celluláris targetjének, az MMP2-nek a szerepét. Új eredményeink közül néhányat emelünk ki. Kimutattuk, hogy hiperlipidemiában a szívben a peroxitrit képződés és ezáltal az MMP-2 aktivitása fokozódik, ami különösen hiperlipidmémiában jelentős, és ezt a folyamatot a prékondíció gátolja. DNA-chip vizsgálattal feltérképeztük hiperlipidémia hatására a génkifejeződés változásait a szívizomban. Kimutattuk, hogy az alacsony mértékű peroxinitrit képződés a stessz adaptáció kiváltásában igen fontos szerepet tölt be, hiszen olyan mechanizmusokat aktivál, melyek az iszkémiás stressz során túlzott mértékű peroxinitrit-MMP aktivitást csökkenti. Leírtuk, hogy nemspecifikus MMP gátlókkal az infarktus területe csökkenthető még hiperlipidémiás állatban is. Humán ApoB100 transzgenetikus eger modelleken megfigyeltük, hogy az oxidatív/nitrozatív stressz oka a hiperkoleszterinémia, és nem a hipertrigliceridémia. Kimutattuk továbbá, hogy a fiziológiás peroxinitrit szint, melyet a szívizom kapszaicin-érzékeny neuronjai szabályoznak, a normális szívizom relaxációt tartja fent. A project futamideje alatt az adott témában összesen 16 nemzetközi cikket (impakt faktor >70) közöltünk. | Ischemic heart disease developing due to hyperlipidemia is the number one killer in civilized societies. The present 4-year project was aiming at exploration of cellular mechanisms underlying stress adaptation of the myocardium, i.e. pre- and postconditioning, focusing on the role of peroxynitrite and its cellular target matrix metalloproteinase-2 (MMP2). Here we emphasize only some of the most important results of the project. We have shown that in hyperlipidemia, myocardial peroxynitrite formation and thereby MMP2 activity is increased, which is attenuated by preconditioning. We have mapped the changes in gene expression due to hyperlipidemia by the use of DNA-microarray assay. We have observed that moderate peroxynitrite formation is necessary to trigger the stress adaptation mechanisms, which in turn will decrease the pathological activation of the peroxynitrite-MMP2 signaling. We have shown that nonspecific MMP inhibitors are able to reduce infarct size even in the presence of hyperlipidemia. In human ApoB-100 transgene mice, we have observed that oxidative/nitrosative stress is due to hypercholesterolemia and not hypertriglyceridemia. Furthermore, we have shown that baseline physiological peroxynitrite formation, which is regulated by myocardial capsaicin-sensitive sensory nerves, plays an important role in the maintenance of normal relaxation of the myocardium. The present project yielded altogether 16 peer-reviewed papers (impact factor >70)

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

Calcium signaling as a mediator of cell energy demand and a trigger to cell death

Calcium signaling is pivotal to a host of physiological pathways. A rise in calcium concentration almost invariably signals an increased cellular energy demand. Consistent with this, calcium signals mediate a number of pathways that together serve to balance energy supply and demand. In pathological states, calcium signals can precipitate mitochondrial injury and cell death, especially when coupled to energy depletion and oxidative or nitrosative stress. This review explores the mechanisms that couple cell signaling pathways to metabolic regulation or to cell death. The significance of these pathways is exemplified by pathological case studies, such as those showing loss of mitochondrial calcium uptake 1 in patients and ischemia/reperfusion injury

Simvastatin promotes cardiac myocyte relaxation in association with phosphorylation of Troponin I

The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins’ myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolaemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca2+]i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser23,24. Statin treatment increased expression of eNOS and Ser1177 phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolin 1 and 3, and increased (P=0.06) NO metabolites, consistent with enhanced NO production. It is well established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance

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)

Autophagy Induced by Ischemic Preconditioning is Essential for Cardioprotection

Based on growing evidence linking autophagy to preconditioning, we tested the hypothesis that autophagy is necessary for cardioprotection conferred by ischemic preconditioning (IPC). We induced IPC with three cycles of 5 min regional ischemia alternating with 5 min reperfusion and assessed the induction of autophagy in mCherry-LC3 transgenic mice by imaging of fluorescent autophagosomes in cryosections. We found a rapid and significant increase in the number of autophagosomes in the risk zone of the preconditioned hearts. In Langendorff-perfused hearts subjected to an IPC protocol of 3 × 5 min ischemia, we also observed an increase in autophagy within 10 min, as assessed by Western blotting for p62 and cadaverine dye binding. To establish the role of autophagy in IPC cardioprotection, we inhibited autophagy with Tat-ATG5K130R, a dominant negative mutation of the autophagy protein Atg5. Cardioprotection by IPC was reduced in rat hearts perfused with recombinant Tat-ATG5K130R. To extend the potential significance of autophagy in cardioprotection, we also assessed three structurally unrelated cardioprotective agents—UTP, diazoxide, and ranolazine—for their ability to induce autophagy in HL-1 cells. We found that all three agents induced autophagy; inhibition of autophagy abolished their protective effect. Taken together, these findings establish autophagy as an end-effector in ischemic and pharmacologic preconditioning