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Radiation-induced heart disease (RIHD) is a potential late side-effect of thoracic radiotherapy resulting in left ventricular hypertrophy (LVH) and fibrosis due to a complex pathomechanism leading to heart failure. Angiotensin-II receptor blockers (ARBs), including losartan, are frequently used to control heart failure of various etiologies. Preclinical evidence is lacking on the anti-remodeling effects of ARBs in RIHD, while the results of clinical studies are controversial. We aimed at investigating the effects of losartan in a rat model of RIHD. Male Sprague-Dawley rats were studied in three groups: (1) control, (2) radiotherapy (RT) only, (3) RT treated with losartan (per os 10 mg/kg/day), and were followed for 1, 3, or 15 weeks. At 15 weeks post-irradiation, losartan alleviated the echocardiographic and histological signs of LVH and fibrosis and reduced the overexpression of chymase, connective tissue growth factor, and transforming growth factor-beta in the myocardium measured by qPCR; likewise, the level of the SMAD2/3 protein determined by Western blot decreased. In both RT groups, the pro-survival phospho-AKT/AKT and the phospho-ERK1,2/ERK1,2 ratios were increased at week 15. The antiremodeling effects of losartan seem to be associated with the repression of chymase and several elements of the TGF-β/SMAD signaling pathway in our RIHD model.

Background Metabolic syndrome (MetS) refers to a cluster of co-existing cardio-metabolic risk factors, including visceral obesity, dyslipidemia, hyperglycemia with insulin resistance, and hypertension. As there is a close link between MetS and cardiovascular diseases, we aimed to investigate the sex-based differences in MetS-associated heart failure (HF) and cardiovascular response to regular exercise training (ET). Methods High-fat diet-fed male and female APOB-100 transgenic (HFD/APOB-100, 3 months) mice were used as MetS models, and age- and sex-matched C57BL/6 wild-type mice on standard diet served as healthy controls (SD/WT). Both the SD/WT and HFD/APOB-100 mice were divided into sedentary and ET groups, the latter running on a treadmill (0.9 km/h) for 45 min 5 times per week for 7 months. At month 9, transthoracic echocardiography was performed to monitor cardiac function and morphology. At the termination of the experiment at month 10, blood was collected for serum low-density lipoprotein (LDL)- and high-density lipoprotein (HDL)-cholesterol measurements and homeostatic assessment model for insulin resistance (HOMA-IR) calculation. Cardiomyocyte hypertrophy and fibrosis were assessed by histology. Left ventricular expressions of selected genes associated with metabolism, inflammation, and stress response were investigated by qPCR. Results Both HFD/APOB-100 males and females developed obesity and hypercholesterolemia; however, only males showed insulin resistance. ET did not change these metabolic parameters. HFD/APOB-100 males showed echocardiographic signs of mild HF with dilated ventricles and thinner walls, whereas females presented the beginning of left ventricular hypertrophy. In response to ET, SD/WT males developed increased left ventricular volumes, whereas females responded with physiologic hypertrophy. Exercise-trained HFD/APOB-100 males presented worsening HF with reduced ejection fraction; however, ET did not change the ejection fraction and reversed the echocardiographic signs of left ventricular hypertrophy in HFD/APOB-100 females. The left ventricular expression of the leptin receptor was higher in females than males in the SD/WT groups. Left ventricular expression levels of stress response-related genes were higher in the exercise-trained HFD/APOB-100 males and exercise-trained SD/WT females than exercise-trained SD/WT males. Conclusions HFD/APOB-100 mice showed sex-specific cardiovascular responses to MetS and ET; however, left ventricular gene expressions were similar between the groups except for leptin receptor and several stress response-related genes., Highlights Both HFD/APOB-100 males and females developed obesity and hypercholesterolemia; however, only males presented insulin resistance. Exercise training did not change these metabolic parameters significantly.HFD/APOB-100 males showed echocardiographic signs of mild heart failure with thinner walls and dilated ventricles, whereas females developed a starting left ventricular hypertrophy assessed by echocardiography and histology.In response to exercise training, SD/WT males developed increased left ventricular volumes, and females presented physiologic hypertrophy.Exercise-trained HFD/APOB-100 males presented worsening heart failure with reduced ejection fraction. On the contrary, exercise-trained HFD/APOB-100 females reversed the echocardiographic signs of left ventricular hypertrophy.Sex, metabolic syndrome, and exercise training alter the gene expression pattern of the myocardium, which may be involved in the development of sex-specific cardiac alterations in the state of metabolic syndrome or to exercise training.

Uremic cardiomyopathy is characterized by diastolic dysfunction (DD), left ventricular hypertrophy (LVH), and fibrosis. Angiotensin-II plays a major role in the development of uremic cardiomyopathy via nitro-oxidative and inflammatory mechanisms. In heart failure, the beta-3 adrenergic receptor (β3-AR) is up-regulated and coupled to endothelial nitric oxide synthase (eNOS)-mediated pathways, exerting antiremodeling effects. We aimed to compare the antiremodeling effects of the angiotensin-II receptor blocker losartan and the β3-AR agonist mirabegron in uremic cardiomyopathy. Chronic kidney disease (CKD) was induced by 5/6th nephrectomy in male Wistar rats. Five weeks later, rats were randomized into four groups: (1) sham-operated, (2) CKD, (3) losartan-treated (10 mg/kg/day) CKD, and (4) mirabegron-treated (10 mg/kg/day) CKD groups. At week 13, echocardiographic, histologic, laboratory, qRT-PCR, and Western blot measurements proved the development of uremic cardiomyopathy with DD, LVH, fibrosis, inflammation, and reduced eNOS levels, which were significantly ameliorated by losartan. However, mirabegron showed a tendency to decrease DD and fibrosis; but eNOS expression remained reduced. In uremic cardiomyopathy, β3-AR, sarcoplasmic reticulum ATPase (SERCA), and phospholamban levels did not change irrespective of treatments. Mirabegron reduced the angiotensin-II receptor 1 expression in uremic cardiomyopathy that might explain its mild antiremodeling effects despite the unchanged expression of the β3-AR.

Chronic obstructive pulmonary disease (COPD) is a common comorbidity of non-small cell lung cancer (NSCLC). COPD is characterized by systemic inflammation and lymphocyte dysfunction, mechanisms that are also known to accelerate progression of advanced (IIIB-IV) stage NSCLC. We aimed to find out whether COPD exerts an influence on tumor induced inflammatory and lymphoid responses and progression-free survival (PFS) after first-line treatment in advanced NSCLC. Patients suffering from NSCLC (n = 95), COPD (n = 54), NSCLC+COPD (n = 80) and healthy controls (n = 60) were included. PFS, neutrophil granulocyte and lymphocyte cell counts were recorded. Serum IFNγ, TNFα, VEGF concentrations were measured by using multiplex cytometric bead-based immunoassay. Prevalence of myeloid-derived suppressor cell populations (MDSC-s), and signs of T cell exhaustion were tested by using flow cytometry. Median PFS increased in the NSCLC+COPD group compared to NSCLC patients without COPD (7.4 vs 4.9 months, p

Hyperlipidemia is a major risk factor associated with increased risk of myocardial infarction. Dipeptidyl peptidase-4 (DPP-4) inhibitors such as sitagliptin are a class of oral anti-diabetic drugs with secondary pleiotropic effects on metabolic and cardiovascular parameters. This study aimed to determine the possible cardioprotective effects of sitagliptin on ischemia-reperfusion (I/R) injury in animals kept on high-fat diet. Male Wistar rats were fed with high-fat diet (HF) for 12 weeks, to induce hyperlipidemia. During the last two weeks of the feeding period, animals were orally treated with different doses of sitagliptin (Sitg: 25, 50, 100, and 150 mg/kg/day), or saline as a control. Heart tissues were then isolated and subjected to two different I/R-injury protocols for infarct size (IS) measurement and biochemical analysis. To test the role of NOS enzyme, NOS inhibitor (L-NAME) was injected intraperitoneally for IS evaluation. As an effective dose, Sitg (50 mg) exhibited a significant impact on IS. NOS activity increased significantly in the Sitg (50 mg) treated groups; however this protective effect was abolished in the presence of L-NAME. The protective effect of Sitg that was mediated by TRP channels in our previous study on normolipidemic animals was abrogated in animals fed with high-fat diet.

Asthma is a chronic disease of the airways, which affects more than 350 million people worldwide. It is the most common chronic disease in children, affecting at least 30 million children and young adults in Europe. Asthma is a complex, partially heritable disease with a marked heterogeneity. Its development is influenced both by genetic and environmental factors. The most common, as well as the most well characterized subtype of asthma is allergic eosinophilic asthma, which is characterized by a type 2 airway inflammation. The prevalence of asthma has substantially increased in industrialized countries during the last 60 years. The mechanisms underpinning this phenomenon are incompletely understood, however increased exposure to various environmental pollutants probably plays a role. Disease inception is thought to be enabled by a disadvantageous shift in the balance between protective and harmful lifestyle and environmental factors, including exposure to protective commensal microbes versus infection with pathogens, collectively leading to airway epithelial cell damage and disrupted barrier integrity. Epithelial cell-derived cytokines are one of the main drivers of the type 2 immune response against innocuous allergens, ultimately leading to infiltration of lung tissue with type 2 T helper (TH2) cells, type 2 innate lymphoid cells (ILC2s), M2 macrophages and eosinophils. This review outlines the mechanisms responsible for the orchestration of type 2 inflammation and summarizes the novel findings, including but not limited to dysregulated epithelial barrier integrity, alarmin release and innate lymphoid cell stimulation.

Inappropriate nutrition and a sedentary lifestyle can lead to obesity, one of the most common risk factors for several chronic diseases. Although regular physical exercise is an efficient approach to improve cardiometabolic health, the exact cellular processes are still not fully understood. We aimed to analyze the morphological, gene expression, and lipidomic patterns in the liver and adipose tissues in response to regular exercise. Healthy (wild type on a normal diet) and hyperlipidemic, high-fat diet-fed (HFD-fed) apolipoprotein B-100 (APOB-100)-overexpressing mice were trained by treadmill running for 7 months. The serum concentrations of triglyceride and tumor necrosis factor α (TNFα), as well as the level of lipid accumulation in the liver, were significantly higher in HFD-fed APOB-100 males compared to females. However, regular exercise almost completely abolished lipid accumulation in the liver of hyperlipidemic animals. The expression level of the thermogenesis marker, uncoupling protein-1 (Ucp1), was significantly higher in the subcutaneous white adipose tissue of healthy females, as well as in the brown adipose tissue of HFD-fed APOB-100 females, compared to males. Lipidomic analyses revealed that hyperlipidemia essentially remodeled the lipidome of brown adipose tissue, affecting both the membrane and storage lipid fractions, which was partially restored by exercise in both sexes. Our results revealed more severe metabolic disturbances in HFD-fed APOB-100 males compared to females. However, exercise efficiently reduced the body weight, serum triglyceride levels, expression of pro-inflammatory factors, and hepatic lipid accumulation in our model.

Introduction Nasal nitric oxide (nNO) measurement can be a useful tool for monitoring upper airway diseases. However, there is a considerable lack of validation data. Aims To evaluate the repeatability and intra-subject variations of nNO output (nVNO) in healthy adults and to study its correlation with lower airway NO parameters. Methods nVNO was measured in healthy non-smokers at baseline (N=31, age: 28±6 years), after 1 hour (N=15), 1 day (N=15), 1 week (N=17), and compared using the Bland-Altman method. At baseline, lower airway NO parameters (FENO, flux of NO in the conducting airways and alveolar NO concentration) were also measured and correlated to nVNO (Spearman correlation). Multivariate regression analysis was used to assess the factors influencing nVNO. Results Baseline median nVNO was 465 (interquartile range=404-536) nL/min. The mean differences between the baseline and repeated measurements were not significant (p>0.05). The coefficient of repeatability (mean: 118, interquartile range=88-181 nL/min) and coefficient of variation (mean: 9.1%) were low. We found no correlation between nVNO and lower airway NO parameters (p>0.05). Sex (β=-0.52, p=0.02) and body weight (β=-0.65, p=0.03) influenced nVNO (model: p=0.04, R2=0.31). Conclusion nNO output has good repeatability in healthy adults. The NO productions of lower and upper airways are not related in health, but nasal NO output seems to be affected by sex and body weight.

Background Lipid accumulation in the liver and pancreas is primarily caused by combined hyperlipidemia. However, the effect of isolated hypercholesterolemia without hypertriglyceridemia is not fully described. Therefore, our aim was to investigate whether hypercholesterolemia alone leads to alterations both in hepatic and pancreatic lipid panel and histology in rats. Methods Male Wistar rats were fed with 2% cholesterol +0.25% cholate-supplemented diet or standard chow for 12 weeks. Blood was collected at weeks 0, 4, 8 and 12 to measure serum cholesterol and triglyceride levels. At week 12, both the pancreas and the liver were isolated for further histological and biochemical analysis. Hepatic and plasma fatty acid composition was assessed by gas chromatography. Expression of mRNA of major enzymes involved in saturated/unsaturated fatty acid synthesis was analyzed by qPCR. In separate experiments serum enzyme activities and insulin levels were measured at week 9. Results At week 12, rats fed with 2% cholesterol +0.25% cholate-supplemented diet were characterized by elevated serum cholesterol (4.09 ± 0.20 vs. 2.89 ± 0.22 mmol/L, *p

Estrogens and raloxifene (RAL) have beneficial effects on certain cardiovascular indices in postmenopausal women characterized by estrogen deficiency. Heme oxygenase (HO) activity is increased by 17β-estradiol (E2) and RAL in estrogen-deficient rat resulting in vasorelaxation mediated by carbon monoxide. We determined the expressions of HO in cardiac and aortic tissues after ovariectomy (OVX) and subsequent RAL or E2treatment. We investigated the effects of pharmacological inhibition of HO enzyme on the arginine vasopressin- (AVP-) induced blood pressure in vivo, the epinephrine- and phentolamine-induced electrocardiogram ST segment changes in vivo, and the myeloperoxidase (MPO) enzyme activity. When compared with intact females, OVX decreased the HO-1 and HO-2 expression, aggravated the electrocardiogram signs of heart ischemia and the blood pressure response to AVP, and increased the cardiac MPO. E2and RAL are largely protected against these negative impacts induced by OVX. The pharmacological inhibition of HO in E2- or RAL-treated OVX animals, however, restored the cardiovascular status close to that observed in nontreated OVX animals. The decreased expression of HO enzymes and the changes in blood pressure ischemia susceptibility and inflammatory state in OVX rat can be reverted by the administration of E2or RAL partly through its antioxidant and anti-inflammatory roles.

Background: Chronic obstructive pulmonary disease (COPD) is related to endothelial dysfunction and the impaired generation of nitric oxide (NO) from L-arginine by the endothelial NO synthase (eNOS). The relationship between eNOS dysfunctionality and airway inflammation is unknown. We assessed serum asymmetric and symmetric dimethylarginine (ADMA and SDMA) and nitrite/nitrate concentrations, indicators of eNOS function, in patients with COPD and correlated them with markers of inflammation. Methods: We recruited 15 control smokers, 29 patients with stable and 32 patients with exacerbated COPD requiring hospitalization (20 of them were measured both at admission and discharge). Serum L-arginine, ADMA, SDMA, nitrite and nitrate were measured and correlated with airway inflammatory markers (fractional exhaled nitric oxide concentration - FENO, sputum nitrite and nitrate, sputum cellularity), serum C-reactive protein - CRP, white blood cell count, lung function and blood gases. ANOVA, t-tests and Pearson correlation were used (mean ± SD or geometric mean ± geometric SD for nitrite/nitrate). Results: Serum L-arginine/ADMA, a marker of substrate availability for eNOS, was lower in stable (214 ± 58, p

Background: A deleterious, late-onset side effect of thoracic radiotherapy is the development of radiation-induced heart disease (RIHD). It covers a spectrum of cardiac pathology including also heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MicroRNA-212 (miR-212) is a crucial regulator of pathologic LVH via FOXO3-mediated pathways in pressure-overload-induced heart failure. We aimed to investigate whether miR-212 and its selected hypertrophy-associated targets play a role in the development of RIHD. Methods: RIHD was induced by selective heart irradiation (50 Gy) in a clinically relevant rat model. One, three, and nineteen weeks after selective heart irradiation, transthoracic echocardiography was performed to monitor cardiac morphology and function. Cardiomyocyte hypertrophy and fibrosis were assessed by histology at week 19. qRT-PCR was performed to measure the gene expression changes of miR-212 and forkhead box O3 (FOXO3) in all follow-up time points. The cardiac transcript level of other selected hypertrophy-associated targets of miR-212 including extracellular signal-regulated kinase 2 (ERK2), myocyte enhancer factor 2a (MEF2a), AMP-activated protein kinase, (AMPK), heat shock protein 40 (HSP40), sirtuin 1, (SIRT1), calcineurin A-alpha and phosphatase and tensin homolog (PTEN) were also measured at week 19. Cardiac expression of FOXO3 and phospho-FOXO3 were investigated at the protein level by Western blot at week 19. Results: In RIHD, diastolic dysfunction was present at every time point. Septal hypertrophy developed at week 3 and a marked LVH with interstitial fibrosis developed at week 19 in the irradiated hearts. In RIHD, cardiac miR-212 was overexpressed at week 3 and 19, and FOXO3 was repressed at the mRNA level only at week 19. In contrast, the total FOXO3 protein level failed to decrease in response to heart irradiation at week 19. Other selected hypertrophy-associated target genes failed to change at the mRNA level in RIHD at week 19. Conclusions: LVH in RIHD was associated with cardiac overexpression of miR-212. However, miR-212 seems to play a role in the development of LVH via FOXO3-independent mechanisms in RIHD. As a central regulator of pathologic remodeling, miR-212 might become a novel target for RIHD-induced LVH and heart failure.

Prediabetes is a condition affecting more than 35% of the population. In some forms, excessive carbohydrate intake (primarily refined sugar) plays a prominent role. Prediabetes is a symptomless, mostly unrecognized disease which increases cardiovascular risk. In our work, we examined the effect of a fructose-enriched diet on cardiac function and lipidome as well as proteome of cardiac muscle. Male Wistar rats were divided into two groups. The control group received a normal diet while the fructose-fed group received 60% fructose-supplemented chow for 24 weeks. Fasting blood glucose measurement and oral glucose tolerance test (OGTT) showed slightly but significantly elevated values due to fructose feeding indicating development of a prediabetic condition. Both echocardiography and isolated working heart perfusion performed at the end of the feeding protocol demonstrated diastolic cardiac dysfunction in the fructose-fed group. Mass spectrometry-based, high-performance lipidomic and proteomic analyses were executed from cardiac tissue. The lipidomic analysis revealed complex rearrangement of the whole lipidome with special emphasis on defects in cardiolipin remodeling. The proteomic analysis showed significant changes in 75 cardiac proteins due to fructose feeding including mitochondria-, apoptosis-, and oxidative stress-related proteins. Nevertheless, just very weak or no signs of apoptosis induction and oxidative stress were detected in the hearts of fructose-fed rats. Our results suggest that fructose feeding induces marked alterations in the cardiac lipidome, especially in cardiolipin remodeling, which leads to mitochondrial dysfunction and impaired cardiac function. However, at the same time, several adaptive responses are induced at the proteome level in order to maintain a homeostatic balance. These findings demonstrate that even very early stages of prediabetes can impair cardiac function and can result in significant changes in the lipidome and proteome of the heart prior to the development of excessive oxidative stress and cell damage.

Animal models have historically played a critical role in the exploration and characterization of disease pathophysiology and target identification and in the evaluation of novel therapeutic agents and treatments in vivo. Diabetes mellitus disease, commonly known as diabetes, is a group of metabolic disorders characterized by high blood glucose levels for a prolonged time. To avoid late complications of diabetes and related costs, primary prevention and early treatment are therefore necessary. Due to its chronic symptoms, new treatment strategies need to be developed, because of the limited effectiveness of the current therapies. We overviewed the pathophysiological features of diabetes in relation to its complications in type 1 and type 2 mice along with rat models, including Zucker Diabetic Fatty (ZDF) rats, BB rats, LEW 1AR1/-iddm rats, Goto-Kakizaki rats, chemically induced diabetic models, and Nonobese Diabetic mouse, and Akita mice model. The advantages and disadvantages that these models comprise were also addressed in this review. This paper briefly reviews the wide pathophysiological and molecular mechanisms associated with type 1 and type 2 diabetes, particularly focusing on the challenges associated with the evaluation and predictive validation of these models as ideal animal models for preclinical assessments and discovering new drugs and therapeutic agents for translational application in humans. The project was subsidized by the European Union and cofinanced by the European Social Fund.

Chronic kidney disease (CKD) is a public health problem and a recognized risk factor for cardiovascular diseases (CVD). CKD could amplify the progression of chronic heart failure leading to the development of type 4 cardio-renal syndrome (T4CRS). The severity and persistence of heart failure are strongly associated with mortality risk in T4CRS. CKD is also a catabolic state leading to renal sarcopenia which is characterized by the loss of skeletal muscle strength and physical function. Renal sarcopenia also promotes the development of CVD and increases the mortality in CKD patients. In turn, heart failure developed in T4CRS could result in chronic muscle hypoperfusion and metabolic disturbances leading to or aggravating the renal sarcopenia. The interplay of multiple factors (e.g., comorbidities, over-activated renin-angiotensin-aldosterone system [RAAS], sympathetic nervous system [SNS], oxidative/nitrative stress, inflammation, etc.) may result in the progression of T4CRS and renal sarcopenia. Among these factors, oxidative/nitrative stress plays a crucial role in the complex pathomechanism and interrelationship between T4CRS and renal sarcopenia. In the heart and skeletal muscle, mitochondria, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, uncoupled nitric oxide synthase (NOS) and xanthine oxidase are major ROS sources producing superoxide anion (O2·−) and/or hydrogen peroxide (H2O2). O2·− reacts with nitric oxide (NO) forming peroxynitrite (ONOO−) which is a highly reactive nitrogen species (RNS). High levels of ROS/RNS cause lipid peroxidation, DNA damage, interacts with both DNA repair enzymes and transcription factors, leads to the oxidation/nitration of key proteins involved in contractility, calcium handling, metabolism, antioxidant defense mechanisms, etc. It also activates the inflammatory response, stress signals inducing cardiac hypertrophy, fibrosis, or cell death via different mechanisms (e.g., apoptosis, necrosis) and dysregulates autophagy. Therefore, the thorough understanding of the mechanisms which lead to perturbations in oxidative/nitrative metabolism and its relationship with pro-inflammatory, hypertrophic, fibrotic, cell death and other pathways would help to develop strategies to counteract systemic and tissue oxidative/nitrative stress in T4CRS and renal sarcopenia. In this review, we also focus on the effects of some well-known and novel pharmaceuticals, nutraceuticals, and physical exercise on cardiac and skeletal muscle oxidative/nitrative stress in T4CRS and renal sarcopenia.

Chronic kidney disease (CKD) is a public health problem that increases the risk of cardiovascular morbidity and mortality. Heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction is a common cardiovascular complication of CKD. MicroRNA-212 (miR-212) has been demonstrated previously to be a crucial regulator of pathologic LVH in pressure-overload-induced heart failure via regulating the forkhead box O3 (FOXO3)/calcineurin/nuclear factor of activated T-cells (NFAT) pathway. Here we aimed to investigate whether miR-212 and its hypertrophy-associated targets including FOXO3, extracellular signal-regulated kinase 2 (ERK2), and AMP-activated protein kinase (AMPK) play a role in the development of HFpEF in CKD. CKD was induced by 5/6 nephrectomy in male Wistar rats. Echocardiography and histology revealed LVH, fibrosis, preserved systolic function, and diastolic dysfunction in the CKD group as compared to sham-operated animals eight and/or nine weeks later. Left ventricular miR-212 was significantly overexpressed in CKD. However, expressions of FOXO3, AMPK, and ERK2 failed to change significantly at the mRNA or protein level. The protein kinase B (AKT)/FOXO3 and AKT/mammalian target of rapamycin (mTOR) pathways are also proposed regulators of LVH induced by pressure-overload. Interestingly, phospho-AKT/total-AKT ratio was increased in CKD without significantly affecting phosphorylation of FOXO3 or mTOR. In summary, cardiac overexpression of miR-212 in CKD failed to affect its previously implicated hypertrophy-associated downstream targets. Thus, the molecular mechanism of the development of LVH in CKD seems to be independent of the FOXO3, ERK1/2, AMPK, and AKT/mTOR-mediated pathways indicating unique features in this form of LVH.

Matrix metalloproteinases (MMP) belong to a distinguished class of zinc-dependent endopeptidases. Zymography is a semi-quantitative tool for determining the activity of different MMP isoenzymes in a variety of biological samples. In substrate gel zymography, protein samples of different origin (tissue, cell lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacrylamide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling substrate cleavage by MMPs, MMP activities are detected after the gel staining as transparent bands against a dark-blue background. In situ zymography is a histological modification of substrate zymography in frozen sections, allowing detection of the localization of the MMP activities within the tissue. Here, we describe detailed experimental protocols of all abovementioned techniques and provide examples for several sample measurements.

Matrix metalloproteinases (MMP) belong to a distinguished class of zinc-dependent endopeptidases. Zymography is a semi-quantitative tool for determining the activity of different MMP isoenzymes in a variety of biological samples. In substrate gel zymography, protein samples of different origin (tissue, cell lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacrylamide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling substrate cleavage by MMPs, MMP activities are detected after the gel staining as transparent bands against a dark-blue background. In situ zymography is a histological modification of substrate zymography in frozen sections, allowing detection of the localization of the MMP activities within the tissue. Here, we describe detailed experimental protocols of all abovementioned techniques and provide examples for several sample measurements.

Diet-induced hypercholesterolemia leads to oxidative/nitrative stress and subsequent myocardial dysfunction. However, the regulatory role of microRNAs in this phenomenon is unknown. We aimed to investigate, whether hypercholesterolemia-induced myocardial microRNA alterations play a role in the development of oxidative/nitrative stress and in subsequent cardiac dysfunction. Male Wistar rats were fed with 2% cholesterol/0.25% cholate-enriched or standard diet for 12weeks. Serum and tissue cholesterol levels were significantly elevated by cholesterol-enriched diet. Left ventricular end-diastolic pressure was significantly increased in cholesterol-fed rats both in vivo and in isolated perfused hearts, indicating diastolic dysfunction. Myocardial expression of microRNAs was affected by cholesterol-enriched diet as assessed by microarray analysis. MicroRNA-25 showed a significant down-regulation as detected by microarray analysis and QRT-PCR. In silico target prediction revealed NADPH oxidase 4 (NOX4) as a putative target of microRNA-25. NOX4 protein showed significant up-regulation in the hearts of cholesterol-fed rats, while NOX1 and NOX2 remained unaffected. Cholesterol-feeding significantly increased myocardial oxidative/nitrative stress as assessed by dihydroethidium staining, protein oxidation assay, and nitro-tyrosine ELISA, respectively. Direct binding of microRNA-25 mimic to the 3' UTR region of NOX4 was demonstrated using a luciferase reporter assay. Transfection of a microRNA-25 mimic into primary cardiomyocytes decreased superoxide production, while a microRNA-25 inhibitor resulted in an up-regulation of NOX4 protein and an increase in oxidative stress that was attenuated by the NADPH oxidase inhibitor diphenyleneiodonium. Here we demonstrated for the first time that hypercholesterolemia affects myocardial microRNA expression, and by down-regulating microRNA-25 increases NOX4 expression and consequently oxidative/nitrative stress in the heart. We conclude that hypercholesterolemia-induced microRNA alterations play an important role in the regulation of oxidative/nitrative stress and in consequent myocardial dysfunction.

Farnesol is a key metabolite of the mevalonate pathway and known as an antioxidant. We examined whether farnesol treatment protects the ischemic heart. Male Wistar rats were treated orally with 0.2, 1, 5, and 50 mg/kg/day farnesol/vehicle for 12 days, respectively. On day 13, the effect of farnesol treatment on cardiac ischemic tolerance and biochemical changes was tested. Therefore, hearts were isolated and subjected either to 30 min coronary occlusion followed by 120 min reperfusion to measure infarct size or to 10 min aerobic perfusion to measure cardiac mevalonate pathway end-products (protein prenylation, cholesterol, coenzyme Q9, coenzyme Q10, dolichol), and 3-nitrotyrosine (oxidative/nitrosative stress marker), respectively. The cytoprotective effect of farnesol was also tested in cardiomyocytes subjected to simulated ischemia/reperfusion. Farnesol pretreatment decreased infarct size in a U-shaped dose–response manner where 1 mg/kg/day dose reached a statistically significant reduction (22.3 ± 3.9 % vs. 40.9 ± 6.1 % of the area at risk, p

Erythropoietin (EPO) has been shown to protect the heart against acute myocardial infarction in pre-clinical studies, however, EPO failed to reduce infarct size in clinical trials and showed significant safety problems. Here, we investigated cardioprotective effects of two selective non-erythropoietic EPO receptor ligand dimeric peptides (AF41676 and AF43136) lacking erythropoietic activity, EPO, and the prolonged half-life EPO analogue, darbepoetin in acute myocardial infarction (AMI) in rats. In a pilot study, EPO at 100 U/mL significantly decreased cell death compared to vehicle (33.8 ± 2.3% vs. 40.3 ± 1.5%, p < 0.05) in rat neonatal cardiomyocytes subjected to simulated ischemia/reperfusion. In further studies (studies 1–4), in vivo AMI was induced by 30 min coronary occlusion and 120 min reperfusion in male Wistar rats. Test compounds and positive controls for model validation (B-type natriuretic peptide, BNP or cyclosporine A, CsA) were administered iv. before the onset of reperfusion. Infarct size (IS) was measured by standard TTC staining. In study 1, 5000 U/kg EPO reduced infarct size significantly compared to vehicle (45.3 ± 4.8% vs. 59.8 ± 4.5%, p < 0.05). In study 2, darbepoetin showed a U-shaped dose-response curve with maximal infarct size-reducing effect at 5 μg/kg compared to the vehicle (44.4 ± 5.7% vs. 65.9 ± 2.7%, p < 0.01). In study 3, AF41676 showed a U-shaped dose-response curve, where 3 mg/kg was the most effective dose compared to the vehicle (24.1 ± 3.9% vs. 44.3 ± 2.5%, p < 0.001). The positive control BNP significantly decreased infarct size in studies 1–3 by approximately 35%. In study 4, AF43136 at 10 mg/kg decreased infarct size, similarly to the positive control CsA compared to the appropriate vehicle (39.4 ± 5.9% vs. 58.1 ± 5.4% and 45.9 ± 2.4% vs. 63.8 ± 4.1%, p < 0.05, respectively). This is the first demonstration that selective, non-erythropoietic EPO receptor ligand dimeric peptides AF41676 and AF43136 administered before reperfusion are able to reduce infarct size in a rat model of AMI. Therefore, non-erythropoietic EPO receptor peptide ligands may be promising cardioprotective agents. Janos Bolyai Research Scholarships of the Hungarian Academy of Sciences. Postdoctoral Research Program of the Hungarian Academy of Sciences. Szentagothai Fellowship (TAMOP-4.2.4.A/2-11/1-2012-0001) of the National Excellence Program of Hungary. Scopus

Additional file 2. Additional Tables.

Dipeptidyl peptidase-4 (DPP-4) inhibitors are a class of oral anti-diabetic drugs, implicated in pleiotropic secondary cardioprotective effects. The aim of the study was to unveil the unknown and possible cardioprotective targets that can be exerted by sitagliptin (Sitg) against ischemia-reperfusion (I/R) injury. Male wistar rats received 2 weeks? Sitg oral treatment of different doses (25, 50, 100, and 150 mg/kg/day), or saline as a Control. Hearts were then isolated and subjected to two different I/R injury protocols: 10 min perfusion, 45 min regional ischemia, and 120 min reperfusion for infarct size (IS) measurement, or: 10 min perfusion, 45 min regional ischemia and 10 min reperfusion for biochemical analysis: nitric oxide synthases (NOSs) and DPP-4 activity, glucagon-like peptide-1 (GLP-1), Calcium, transient receptor potential vanilloid (TRPV)-1 and calcitonin gene-related peptide (CGRP) levels, transient receptor potential canonical (TRPC)-1 and e-NOS protein expression. NOS inhibitor (L-NAME) and TRPV-1 inhibitor (Capsazepine) were utilized to confirm the implication of both signaling mechanisms in DPP-4 inhibition-induced at the level of IS. Findings show that Sitg (50 mg) resulted in significant decrease in IS and DPP-4 activity, and significant increase in GLP-1, NOS activity, e-NOS expression, TRPV-1 level and TRPC-1 expression, compared to controls. Results of CGRP are in line with TRPV-1, as a downstream regulatory effect. NOS system and transient receptor potential (TRP) channels can contribute to DPP-4 inhibition-mediated cardioprotection against I/R injury using Sitagliptin. This research was funded by GINOP grant number 2.3.2-15-2016-00035. Scopus

Background Although complex multivitamin products are widely used as dietary supplements to maintain health or as special medical food in certain diseases, the effects of these products were not investigated in hyperlipidemia which is a major risk factor for cardiovascular diseases. Therefore, here we investigated if a preparation developed for human use containing different vitamins, minerals and trace elements enriched with phytosterol (VMTP) affects the severity of experimental hyperlipidemia as well as myocardial ischemia/reperfusion injury. Methods Male Wistar rats were fed a normal or cholesterol-enriched (2% cholesterol + 0.25% cholate) diet for 12 weeks to induce hyperlipidemia. From week 8, rats in both groups were fed with a VMTP preparation or placebo for 4 weeks. Serum triglyceride and cholesterol levels were measured at week 0, 8 and 12. At week 12, hearts were isolated, perfused according to Langendorff and subjected to a 30-min coronary occlusion followed by 120 min reperfusion to measure infarct size. Results At week 8, cholesterol-fed rats showed significantly higher serum cholesterol level as compared to normal animals, however, serum triglyceride level did not change. VMTP treatment significantly decreased serum cholesterol level in the hyperlipidemic group by week 12 without affecting triglyceride levels. However, VMTP did not show beneficial effect on infarct size. The inflammatory marker hs-CRP and the antioxidant uric acid were also not significantly different. Conclusions This is the first demonstration that treatment of hyperlipidemic subjects with a VMTP preparation reduces serum cholesterol, the major risk factor for cardiovascular disease; however, it does not provide cardioprotection.

Background Metabolic syndrome (coexisting visceral obesity, dyslipidemia, hyperglycemia, and hypertension) is a prominent risk factor for cardiovascular morbidity and mortality, however, its effect on cardiac gene expression pattern is unclear. Therefore, we examined the possible alterations in cardiac gene expression pattern in male Zucker Diabetic Fatty (ZDF) rats, a model of metabolic syndrome. Methods Fasting blood glucose, serum insulin, cholesterol and triglyceride levels were measured at 6, 16, and 25 wk of age in male ZDF and lean control rats. Oral glucose tolerance test was performed at 16 and 25 wk of age. At week 25, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 14921 genes. Expression of selected genes was confirmed by qRT-PCR. Results Fasting blood glucose, serum insulin, cholesterol and triglyceride levels were significantly increased, glucose tolerance and insulin sensitivity were impaired in ZDF rats compared to leans. In hearts of ZDF rats, 36 genes showed significant up-regulation and 49 genes showed down-regulation as compared to lean controls. Genes with significantly altered expression in the heart due to metabolic syndrome includes functional clusters of metabolism (e.g. 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2; argininosuccinate synthetase; 2-amino-3-ketobutyrate-coenzyme A ligase), structural proteins (e.g. myosin IXA; aggrecan1), signal transduction (e.g. activating transcription factor 3; phospholipase A2; insulin responsive sequence DNA binding protein-1) stress response (e.g. heat shock 70kD protein 1A; heat shock protein 60; glutathione S-transferase Yc2 subunit), ion channels and receptors (e.g. ATPase, (Na+)/K+ transporting, beta 4 polypeptide; ATPase, H+/K+ transporting, nongastric, alpha polypeptide). Moreover some other genes with no definite functional clusters were also changed such as e.g. S100 calcium binding protein A3; ubiquitin carboxy-terminal hydrolase L1; interleukin 18. Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by metabolic syndrome. Conclusions Metabolic syndrome significantly alters cardiac gene expression profile which may be involved in development of cardiac pathologies in the presence of metabolic syndrome.

Background Diabetic patients have an increased risk of developing cardiovascular diseases, which are the leading cause of death in developed countries. Although multivitamin products are widely used as dietary supplements, the effects of these products have not been investigated in the diabetic heart yet. Therefore, here we investigated if a preparation of different minerals, vitamins, and trace elements (MVT) affects the cardiac gene expression pattern in experimental diabetes. Methods Two-day old male Wistar rats were injected with streptozotocin (i.p. 100 mg/kg) or citrate buffer to induce diabetes. From weeks 4 to 12, rats were fed with a vehicle or a MVT preparation. Fasting blood glucose measurement and oral glucose tolerance test were performed at week 12, and then total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41012 oligonucleotides. Results Significantly elevated fasting blood glucose concentration and impaired glucose tolerance were markedly improved by MVT-treatment in diabetic rats at week 12. Genes with significantly altered expression due to diabetes include functional clusters related to cardiac hypertrophy (e.g. caspase recruitment domain family, member 9; cytochrome P450, family 26, subfamily B, polypeptide; FXYD domain containing ion transport regulator 3), stress response (e.g. metallothionein 1a; metallothionein 2a; interleukin-6 receptor; heme oxygenase (decycling) 1; and glutathione S-transferase, theta 3), and hormones associated with insulin resistance (e.g. resistin; FK506 binding protein 5; galanin/GMAP prepropeptide). Moreover the expression of some other genes with no definite cardiac function was also changed such as e.g. similar to apolipoprotein L2; brain expressed X-linked 1; prostaglandin b2 synthase (brain). MVT-treatment in diabetic rats showed opposite gene expression changes in the cases of 19 genes associated with diabetic cardiomyopathy. In healthy hearts, MVT-treatment resulted in cardiac gene expression changes mostly related to immune response (e.g. complement factor B; complement component 4a; interferon regulatory factor 7; hepcidin). Conclusions MVT-treatment improved diagnostic markers of diabetes. This is the first demonstration that MVT-treatment significantly alters cardiac gene expression profile in both control and diabetic rats. Our results and further studies exploring the mechanistic role of individual genes may contribute to the prevention or diagnosis of cardiac complications in diabetes. Electronic supplementary material The online version of this article (doi:10.1186/s12933-015-0248-6) contains supplementary material, which is available to authorized users.