Monika Bartekova

Chronic cardiotoxicity of doxorubicin involves activation of myocardial and circulating matrix metalloproteinases in rats

Aim:To investigate the role of matrix metalloproteinases (MMPs) in the responses of rats to a prolonged doxorubicin (DOX) treatment.Methods:Male Wistar rats were used. DOX was administered by intraperitoneal injections of seven doses (cumulative dose was 15 mg/kg). Control animals were treated with saline. Tissue or plasma samples were collected at four and eight weeks after the application of the last dose. Protein levels were determined by immunoblot assay, and MMP activities were measured by gelatin zymography. Superoxide content was analyzed using a lucigenin chemiluminescence assay and superoxide dismutase (SOD) activities with a SOD assay kit. Qualitative structural alterations of the heart were characterized by transmission electron microscopy.Results:Systolic blood pressure was higher in DOX-treated rats as compared with the control rats at 8 weeks after treatment. In contrast, there were no differences in the heart rate between the control and DOX-treated rats. DOX treatment caused marked heterogeneous subcellular alterations of cardiomyocytes and structural disorganizations of the cardiac extracellular space. The effects of DOX were linked to a stimulation of plasma MMP-2 and MMP-9 activities that had already increased by 4 weeks after the end of the treatment. In the left ventricle, however, DOX only led to increased MMP-2 activation at 8 weeks after the end of treatment. These changes in tissue MMP-2 were connected with stimulation of Akt kinase activation, inhibition of SOD, an increase in superoxide levels, induction of iNOS protein expression and caspase-3 activation.Conclusion:Our results show that MMPs are involved in the chronic cardiotoxicity of DOX in rats. The data also suggest that reactive oxygen species (superoxide), NO production (iNOS) and the Akt kinase pathway can modulate MMP-2 activities in rat hearts influenced by DOX.

Quercetin Improves Postischemic Recovery of Heart Function in Doxorubicin-Treated Rats and Prevents Doxorubicin-Induced Matrix Metalloproteinase-2 Activation and Apoptosis Induction

Quercetin (QCT) is flavonoid that possesses various biological functions including anti-oxidative and radical-scavenging activities. Moreover, QCT exerts some preventive actions in treatment of cardiovascular diseases. The aim of present study was to explore effects of prolonged administration of QCT on changes induced by repeated application of doxorubicin (DOX) in rat hearts. We focused on the ultrastructure of myocardium, matrix metalloproteinases (MMPs), biometric parameters, and apoptosis induction. Our aim was also to examine effects of QCT on ischemic tolerance in hearts exposed to chronic effects of DOX, and to determine possible mechanisms underlying effects of QCT. Our results showed that QCT prevented several negative chronic effects of DOX: (I) reversed DOX-induced blood pressure increase; (II) mediated improvement of deleterious effects of DOX on ultrastructure of left ventricle; (III) prevented DOX-induced effects on tissue MMP-2 activation; and (iv) reversed effects of DOX on apoptosis induction and superoxide dismutase inhibition. Moreover, we showed that rat hearts exposed to effects of QCT were more resistant to ischemia/reperfusion injury. Effects of QCT on modulation of ischemic tolerance were linked to Akt kinase activation and connexin-43 up-regulation. Taken together, these results demonstrate that prolonged treatment with QCT prevented negative chronic effects of DOX on blood pressure, cellular damage, MMP-2 activation, and apoptosis induction. Moreover, QCT influenced myocardial responses to acute ischemic stress. These facts bring new insights into mechanisms of QCT action on rat hearts exposed to the chronic effects of DOX.

Diastolic dysfunction in prediabetic male rats: Role of mitochondrial oxidative stress

Although incidence and prevalence of prediabetes are increasing, little is known about its cardiac effects. Therefore, our aim was to investigate the effect of prediabetes on cardiac function and to characterize parameters and pathways associated with deteriorated cardiac performance. Long-Evans rats were fed with either control or high-fat chow for 21 wk and treated with a single low dose (20 mg/kg) of streptozotocin at week 4 High-fat and streptozotocin treatment induced prediabetes as characterized by slightly elevated fasting blood glucose, impaired glucose and insulin tolerance, increased visceral adipose tissue and plasma leptin levels, as well as sensory neuropathy. In prediabetic animals, a mild diastolic dysfunction was observed, the number of myocardial lipid droplets increased, and left ventricular mass and wall thickness were elevated; however, no molecular sign of fibrosis or cardiac hypertrophy was shown. In prediabetes, production of reactive oxygen species was elevated in subsarcolemmal mitochondria. Expression of mitofusin-2 was increased, while the phosphorylation of phospholamban and expression of Bcl-2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3, a marker of mitophagy) decreased. However, expression of other markers of cardiac auto- and mitophagy, mitochondrial dynamics, inflammation, heat shock proteins, Ca2+/calmodulin-dependent protein kinase II, mammalian target of rapamycin, or apoptotic pathways were unchanged in prediabetes. This is the first comprehensive analysis of cardiac effects of prediabetes indicating that mild diastolic dysfunction and cardiac hypertrophy are multifactorial phenomena that are associated with early changes in mitophagy, cardiac lipid accumulation, and elevated oxidative stress and that prediabetes-induced oxidative stress originates from the subsarcolemmal mitochondria.

Extracellular Vesicles in Cardiovascular Theranostics

Extracellular vesicles (EVs) are small bilayer lipid membrane vesicles that can be released by most cell types and detected in most body fluids. EVs exert key functions for intercellular communication via transferring their bioactive cargos to recipient cells or activating signaling pathways in target cells. Increasing evidence has shown the important regulatory effects of EVs in cardiovascular diseases (CVDs). EVs secreted by cardiomyocytes, endothelial cells, fibroblasts, and stem cells play essential roles in pathophysiological processes such as cardiac hypertrophy, cardiomyocyte survival and apoptosis, cardiac fibrosis, and angiogenesis in relation to CVDs. In this review, we will first outline the current knowledge about the physical characteristics, biological contents, and isolation methods of EVs. We will then focus on the functional roles of cardiovascular EVs and their pathophysiological effects in CVDs, as well as summarize the potential of EVs as therapeutic agents and biomarkers for CVDs. Finally, we will discuss the specific application of EVs as a novel drug delivery system and the utility of EVs in the field of regenerative medicine.

Effect of crowding stress on tolerance to ischemia-reperfusion injury in young male and female hypertensive rats: molecular mechanisms.

Sex and social stress may represent risk factors in the etiology of hypertension and heart response to ischemia-reperfusion (I/R) injury. Phosphatidylinositol 3-kinase/protein kinase B (Akt) plays an important role in the processes associated with hypertension and myocardial tolerance to I/R, and may be involved in myocardial stress reaction. The impact of chronic stress on the response to I/R was investigated in the hearts of 7-week-old spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats of both sexes. Stress was induced by reducing living space to 70 cm(2)/100 g body mass of rat for 2 weeks, while the controls were kept at 200 cm(2)/100 g. Langendorff-perfused hearts, subjected to I/R, exhibited higher vulnerability to ventricular tachycardia in crowd-stressed SHR vs. the control rats, and this was more pronounced in the males. Myocardial infarction was not affected by crowding stress in any of the groups. Male and female SHR showed increased activation of cardiac Akt, whereas nitric oxide synthase activity (NOS) with pro-apoptotic signaling decreased in the males but was not altered in the females (vs. WKY rats). NOS was enhanced in the female SHR and WKY groups by comparison with the respective males. Stress only reduced NOS activity in the SHR groups, and without changes in apoptotic markers. In conclusion, we showed that stress in young SHR mainly affects the nonlethal markers for I/R, and has no impact on myocardial infarction and apoptosis, despite reduced NOS activity.

Pleiotropic preconditioning-like cardioprotective effects of hypolipidemic drugs in acute ischemia-reperfusion in normal and hypertensive rats.

Although pleiotropy, which is defined as multiple effects derived from a single gene, was recognized many years ago, and considerable progress has since been achieved in this field, it is not very clear how much this feature of a drug is clinically relevant. During the last decade, beneficial pleiotropic effects from hypolipidemic drugs (as in, effects that are different from the primary ones) have been associated with reduction of cardiovascular risk. As with statins, the agonists of peroxisome proliferator-activated receptors (PPARs), niacin and fibrates, have been suggested to exhibit pleiotropic activity that could significantly modify the outcome of a cardiovascular ailment. This review examines findings demonstrating the impacts of treatment with hypolipidemic drugs on cardiac response to ischemia in a setting of acute ischemia-reperfusion, in relation to PPAR activation. Specifically, it addresses the issue of susceptibility to ischemia, with particular regard to the preconditioning-like cardioprotection conferred by hypolipidemic drugs, as well as the potential molecular mechanisms behind this cardioprotection. Finally, the involvement of PPAR activation in the mechanisms of non-metabolic cardioprotective effects from hypolipidemic drugs, and their effects on normal and pathologically altered myocardium (in the hearts of hypertensive rats) is also discussed.

Beneficial effects of N-acetylcysteine and N-mercaptopropionylglycine on ischemia reperfusion injury in the heart

BACKGROUND Ischemia-reperfusion (I/R) injury of the heart as a consequence of myocardial infarction or cardiac surgery represents a serious clinical problem. One of the most prominent mechanisms of I/R injury is the development of oxidative stress in the heart. In this regard, I/R has been shown to enhance the production of reactive oxygen/nitrogen species in the heart which lead to the imbalance between the pro-oxidants and antioxidant capacities of the endogenous radical-scavenging systems. OBJECTIVES Increasing the antioxidant capacity of the heart by the administration of exogenous antioxidants is considered beneficial for the heart exposed to I/R. N-acetylcysteine (NAC) and Nmercaptopropionylglycine (MPG) are two sulphur containing amino acid substances, which belong to the broad category of exogenous antioxidants that have been tested for their protective potential in cardiac I/R injury. OBSERVATIONS Pretreatment of hearts with both NAC and MPG has demonstrated that these agents attenuate the I/R-induced alterations in sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils in addition to improving cardiac function. While experimental studies have revealed promising data suggesting beneficial effects of NAC and MPG in cardiac I/R injury, the results of clinical trials are not conclusive because both positive and no effects of these substances have been reported on the post-ischemic recovery of heart following cardiac surgery or myocardial infarction. CONCLUSION It is concluded that both NAC and MPG exert beneficial effects in preventing the I/Rinduced injury; however, further studies are needed to establish their effectiveness in reversing the I/R-induced abnormalities in the heart.

Therapeutic Potential of Hematopoietic Stem Cell-Derived Exosomes in Cardiovascular Disease

As other stem cells, hematopoietic stem cells (HSCs) are able to produce extracellular vesicles (EVs) including exosomes and microvesicles. This chapter summarizes the knowledge about the production of EVs by the HSCs, their role in the intercellular communication, and will discuss the cargo of these EVs as well as protective effects of HSCs-derived exosomes and microvesicles in cardiovascular diseases (CVD). Available data showed that cardioprotective action of injected HSCs could not be explained by direct transdifferentiationof injected cells into the cardiomyocytes, this effect is suggested to be mediated via paracrine communication (by EVs) between donor and recipient cells. Among the cargo molecules of HSCs-derived vesicles several miRNAs, and pro-angiogenic and anti-apoptotic proteins are proposed to be the mediators of heart regeneration, mostly via neovascularization. However, the direct evidence of cardioprotective effects of HSCs-derived exosomes and microvesicles is still lacking in the literature. On the other hand, EVs produced in HSCs-derived cells, specifically dendritic cells and endothelial progenitor cells, have been shown to provide direct cardioprotective effects in CVD. Anyway, further studies are needed to be performed to assess the therapeutic potential of HSCs-derived EVs-based cardiac regenerative therapies.

Effect of yeast biomass with high content of carotenoids on erythrocyte deformability, NO production and Na,K-ATPase activity in healthy and LPS treated rats

Measurements of red blood cell (RBC) deformability together with estimation of NO-synthase activity and Na,K-ATPase activity were used for characterization of RBC functionality in rats subjected to single dose of Escherichia coli lipopolysaccharides (LPS) at a dose of 1 mg/kg. We hypothesized that LPS might initiate a malfunction of RBC. We also investigated the potential effect of carotenoids (10 mg/kg/day) produced in red yeast biomass of Rhodotorula glutinis on RBC in LPS-challenged rats. LPS significantly reduced the deformability of RBC (by 14%) together with decrease of NO-synthase activity by 20%. Daily supplementation of carotenoids for 10 days attenuated the LPS-induced injury, as observed by 22% increase of RBC deformability and 23% increase of NO-synthase activity. The activity of Na,K-ATPase was also improved probably due to increased number of active enzyme molecules as indicated by 66% enhancement of Vmax value, hence maintaining the activity of erythrocyte Na,K-ATPase to the level even higher as compared with healthy control animals. It may be concluded that administration of yeast biomass with high content of carotenoids resulted in advanced function of erythrocytes as concerns their ability to squeeze through narrow capillaries of the circulation, better intrinsic production of NO and improvement of intracellular homeostasis of sodium.

Molecular changes induced by repeated restraint stress in the heart: the effect of oxytocin receptor antagonist atosiban.

Even though stress belongs to the most common lifestyle risk factors of cardiovascular diseases, there are only limited data on direct influence of stressors on the heart. The aim of the present study was to explore selected protein signaling pathways in response to repeated immobilization stress in the heart tissue. Effects of simultaneous treatment with atosiban, an oxytocin receptor antagonist, on stress-induced changes in the heart were also investigated. Male Wistar rats were exposed to repeated immobilization (2 h daily, lasting 2 weeks). The results showed increased phosphorylation of Akt kinase, enhanced levels of Bcl-2, and decreased levels of cleaved caspase-3 in the left ventricle in response to chronic stress independently of the treatment. Exposure to restraint led to the rise of HSP-90 and p53 in vehicle-treated rats only. Stress failed to modify MMP-2 activity and ultrastructure of the heart tissue. Treatment with the oxytocin/vasopressin receptor antagonist atosiban reversed stress-induced rise in HSP-90 and p53 proteins. In conclusion, our data demonstrate that repeated restraint stress induces Akt kinase activation and this is associated with elevation of anti-apoptotic proteins (Bcl-2) and down-regulation of pro-apoptotic proteins (cleaved caspase-3). These findings suggest that activation of pro-survival anti-apoptotic Akt kinase pathway plays an important role in molecular mechanisms underlying responses and adaptation of the rat heart to repeated stress exposure. The results further indicate a regulatory role of oxytocin/vasopressin in the control of stress-induced activation in HSP-90 and related proteins.