Non-Hereditary Obesity Type Networks and New Drug Targets: An In Silico Approach.

Obesity, a chronic, preventable disease, has significant comorbidities that are associated with a great human and financial cost for society. The aim of the present work is to reconstruct the interactomes of non-hereditary obesity to highlight recent advances of its pathogenesis, and discover potential therapeutic targets. Obesity and biological-clock-related genes and/or gene products were extracted from the biomedical literature databases PubMed, GeneCards and OMIM. Their interactions were investigated using STRING v11.0 (a database of known and predicted physical and indirect associations among genes/proteins), and a high confidence interaction score of >0.7 was set. We also applied virtual screening to discover natural compounds targeting obesity- and circadian-clock-associated proteins. Two updated and comprehensive interactomes, the (a) stress- and (b) inflammation-induced obesidomes involving 85 and 93 gene/gene products of known and/or predicted interactions with an average node degree of 9.41 and 10.8, respectively, were produced. Moreover, 15 of these were common between the two non-hereditary entities, namely, ADIPOQ, ADRB2/3, CCK, CRH, CXCL8, FOS, GCG, GNRH1, IGF1, INS, LEP, MC4R, NPY and POMC, while phelligridin E, a natural product, may function as a potent FOX1-DBD interaction blocker. Molecular networks may contribute to the understanding of the integrated regulation of energy balance/obesity pathogenesis and may associate chronopharmacology schemes with natural products.

Epicardial Adipose Tissue in Myocardial Disease: From Physiology to Heart Failure Phenotypes.

Epicardial adipose tissue (EAT) is increasingly being recognized as a determinant of myocardial biology. The EAT-heart crosstalk suggests causal links between dysfunctional EAT and cardiomyocyte impairment. Obesity promotes EAT dysfunction and shifts in secreted adipokines which adversely affect cardiac metabolism, induce cardiomyocyte inflammation, redox imbalance and myocardial fibrosis. Thus, EAT determines cardiac phenotype via effects on cardiac energetics, contractility, diastolic function, and atrial conduction. Vice-versa the EAT is altered in heart failure (HF), and such phenotypic changes can be detected by noninvasive imaging or incorporated in Artificial Intelligence-enhanced tools to aid the diagnosis, subtyping or risk prognostication of HF. In the present article, we summarize the links between EAT and the heart, explaining how the study of epicardial adiposity can improve the understanding of cardiac disease, serve as a source of diagnostic and prognostic biomarkers, and as a potential therapeutic target in HF to improve clinical outcomes.

Tissue and Serum Biomarkers in Degenerative Aortic Stenosis-Insights into Pathogenesis, Prevention and Therapy.

BACKGROUND AND AIM: Degenerative Aortic Stenosis (DAS) is a common disease that causes substantial morbidity and mortality worldwide, especially in the older population. Our aim was to further investigate novel serum and tissue biomarkers to elucidate biological processes involved in this entity. MATERIAL AND METHODS: We evaluated the expression of six biomarkers significantly involved in cardiovascular pathology, i.e., irisin, periostin, osteoglycin, interleukin 18, high mobility group box 1 and proprotein convertase subtilisin/kexin type 9 in the serum at the protein level, and in the tissue at both the protein and mRNA levels of patients with AS (N = 60). Five normal valves obtained after transplantation from hearts of patients with idiopathic dilated cardiomyopathy were also studied. Serum measurements were also performed in 22 individuals without valvular disease who served as controls (C). RESULTS: Higher levels of all factors were found in DAS patients' serum than in normal C. IHC and PCR mRNA tissue analysis showed the presence of all biomarkers in the aortic valve cusps with DAS, but no trace of PCR mRNA was found in the five transplantation valves. Moreover, periostin serum levels correlated significantly with IHC and mRNA tissue levels in AS patients. CONCLUSION: We showed that six widely prevalent biomarkers affecting the atherosclerotic process were also involved in DAS, suggesting a strong osteogenic and pro-inflammatory profile, indicating that aortic valve calcification is a multifactorial biological process.

Neuroendocrine System Adaptation during Consecutive Extrinsic Stimuli: A Pilot Dynamic Study.

This pilot repeated measures study aims to evaluate the dynamics of the autonomic nervous system (ANS), the hypothalamic-pituitary-adrenal (HPA) axis, and/or their interplay with low-level inflammation in healthy schoolchildren during consecutive extrinsic stimuli. Twenty healthy schoolchildren and adolescents aged 11-14 years (12.5 ± 1.5) were consecutively exposed to an oral task (#2) and an arithmetic task (#3) (Trier Social Stress Test for Children (TSST-C)), lasting 5 min each, and a three-minute cellular phone call (#4). Salivary cortisol (SC) was sampled at baseline (#1) and immediately after each exposure (#2, 3, and 4). Baseline serum high-sensitivity C-reactive protein (hsCRP) and cortisol levels were also assessed. ANS dynamics and complexity were measured using Sample Entropy (SampEn) at each experimental time period (#1-4). Baseline serum hCRP and cortisol correlated negatively to each other, while the ANS and HPA axis acute reactions to the three consecutive stimuli differed over time. The ANS adaptation to these stimuli included complexity modulation, which was not dependent on baseline hsCRP or cortisol, and weakened during the third stimulation. However, baseline hsCRP and cortisol had a weakening and an increasing effect on the HPA axis over time, respectively. We conclude that low-level inflammation and baseline morning cortisol level have no effect on ANS dynamics but influence the HPA axis response to consecutive external stimuli.

Peak exercise myocardial deformation indices during cardiopulmonary exercise testing are associated with exercise capacity and ventilatory efficiency in patients with dilated cardiomyopathy.

OBJECTIVE: Little is known about the exercise-induced changes in the multidimensional mechanical properties of the heart. We aimed to evaluate the myocardial deformation indices (MDI) at rest and their response at peak exercise during the same cardiopulmonary exercise testing (CPET) session, investigating their relationship to exercise capacity and ventilatory sufficiency in dilated cardiomyopathy (DCM) patients. METHODS: We evaluated left ventricular (LV) function using speckle tracking imaging (STI) at rest and peak exercise during the same CPET session in 57 idiopathic DCM patients in New York Heart Association (NYHA) I-II class [54 ± 12 years, 42 males, ejection fraction (EF) 33 ± 9%]. We measured global longitudinal strain (GLS), longitudinal strain rate at systole (LSRS) and diastole (LSRD), and circumferential strain rate (CircS). RESULTS: Resting GLS, LSRS, and LSRD were impaired compared with the predicted values but were improved at peak exercise (p < 0.001). All MDI at rest and/or at peak exercise were related to several CPET-derived parameters, including peak VO2, load, O2 pulse, and VE/VCO2 slope. Peak exercise LSRS > -1.10 sec-1 (AUC = 0.80, p < 0.001) and GLS > -13% (AUC = 0.81, p = 0.002) predicted impaired exercise capacity (peak VO2 < 20 ml/min/kg) and ventilatory inefficiency (VE/VCO2 slope>34). In multiple regression analysis, peak exercise LSRS and GLS were independently related to the peak VO2 (Beta = -0.39, p = 0.003) and VE/VCO2 slope (Beta = 0.35, p = 0.02), respectively. CONCLUSIONS: Peak exercise LSRS and GLS in NYHA I-II DCM patients subjected to CPET were associated with aerobic exercise capacity and ventilatory efficiency. Consequently, LSRS and GLS at peak exercise, through their association with CPET-derived CV risk indices, may underline the severity of heart failure and predict future CV events in this DCM population.

Hypertension Predisposition and Thermoregulation Delays in Adolescents with Polycystic Ovary Syndrome: A Pilot Study.

BACKGROUND: Polycystic ovary syndrome (PCOS) is a heterogeneous disorder in which clinical, sonographic, and endophenotypic features have been underinvestigated or inconclusive, especially in the early stages of the disease (adolescence/young adulthood). OBJECTIVE: This prospective pilot study focused on the differences of multiple physiological functions between Greek adolescent/young adult females suffering from PCOS and age- and body mass index (BMI)- matched healthy controls. STUDY DESIGN: Nineteen PCOS patients and eighteen healthy controls (aged 13 to 23 years) were studied for: (i) biochemical and hormonal dysfunction by measuring circulating glucose, insulin, and androgen levels; (ii) arterial stiffness with pulse wave analysis (PWA) by Sphygmocord; (iii) intima-media thickness (IMT) by ultrasound; (iv) heart rate variability (HRV) by Task Force Monitor; and (v) QT, QRS, QT, P, QRSD by electrocardiogram (ECG). Statistical analysis included Hedge's g correction for small samples bias, and the results are shown using the Hedge's g effect size and 95% CI, in line with precision medicine prerequisites. RESULTS: Significant differences in pulse wave velocity (PWV) (g = 0.964 [0.296, 1.632]), subendocardial viability ratio (SEVR) carotid (g = -0.679 [-1.329, -0.030]), pulse pressure (PP) carotid (g = 0.942 [0.275, 1.608]), systolic pressure (SP) carotid (g = 0.785 [0.129, 1.440]), free-testosterone (g = 0.677 [0.042, 0.312]), and Delta4-androstenedione (g = 0.735 [0.097, 0.373]) were observed between PCOS patients and controls. No differences were detected in the remaining endocrine and PWA or ECG biomarkers. CONCLUSIONS: Our multidisciplinary approach showed early onset of vascular dysfunction, predisposition to hypertension, thermoregulation delays, and metabolic syndrome changes in adolescent/young adult PCOS.

A High-Cholesterol Diet Increases Toll-like Receptors and Other Harmful Factors in the Rabbit Myocardium: The Beneficial Effect of Statins.

BACKGROUND: A high-cholesterol diet (HCD) induces vascular atherosclerosis through vascular inflammatory and immunological processes via TLRs. The aim of this study is to investigate the mRNA expression of TLRs and other noxious biomarkers expressing inflammation, fibrosis, apoptosis, and cardiac dysfunction in the rabbit myocardium during (a) high-cholesterol diet (HCD), (b) normal diet resumption and (c) fluvastatin or rosuvastatin treatment. METHODS: Forty-eight male rabbits were randomly divided into eight groups (n = 6/group). In the first experiment, three groups were fed with HCD for 1, 2 and 3 months. In the second experiment, three groups were fed with HCD for 3 months, followed by normal chow for 1 month and administration of fluvastatin or rosuvastatin for 1 month. Control groups were fed with normal chow for 90 and 120 days. The whole myocardium was removed; total RNA was isolated from acquired samples, and polymerase chain reaction, reverse transcription PCR and quantitative real-time PCR were performed. RESULTS: mRNA of TLRs 2, 3, 4 and 8; interleukin-6; TNF-a; metalloproteinase-2; tissue inhibitor of metalloproteinase-1; tumor protein 53; cysteinyl aspartate specific proteinase-3; and brain natriuretic peptide (BNP) increased in HCD. Statins but not resumption of a normal diet decreased levels of these biomarkers and increased levels of antifibrotic factors. CONCLUSIONS: HCD increases the levels of TLRs; inflammatory, fibrotic and apoptotic factors; and BNP in the rabbit myocardium. Atherogenic diets adversely affect the myocardium at a molecular level and are reversed by statins.

Interactions Networks for Primary Heart Sarcomas.

Personalized medicine incorporates genetic information into medical practice so as to optimize the management of chronic diseases. In rare diseases, such as heart cancer (incidence 0.0017-0.33%), this may be elusive. Ninety-five percent of the cases are due to secondary involvementwith the neoplasm originating in the lungs, breasts, kidney, blood, or skin. The clinical manifestations of heart tumors (benign or malignant) include heart failure, hypertension, and cardiac arrhythmias of varying severity, frequently resulting in blood vessel emboli, including strokes. This study aims to explain the pathophysiology and contribute to a P4 medicine model for use by cardiologists, pathologists, and oncologists. We created six gene/protein heart-related and tumor-related targets high-confidence interactomes, which unfold the main pathways that may lead to cardiac diseases (heart failure, hypertension, coronary artery disease, arrhythmias), i.e., the sympathetic nervous system, the renin-angiotensin-aldosterone axis and the endothelin pathway, and excludes others, such as the K oxidase or cytochrome P450 pathways. We concluded that heart cancer patients could be affected by beta-adrenergic blockers, ACE inhibitors, QT-prolonging antiarrhythmic drugs, antibiotics, and antipsychotics. Interactomes may elucidate unknown pathways, adding to patient/survivor wellness during/after chemo- and/or radio-therapy.

Polycystic οvary syndrome revisited: An interactions network approach.

BACKGROUND: The polycystic ovary syndrome (PCOS) has genetic, epigenetic, metabolic and reproductive aspects, while its complex pathophysiology has not been conclusively deciphered. AIM: The goal of this research was to screen the gene/gene products associated with PCOS and to predict any possible interactions with the highest possible fidelity. MATERIALS AND METHODS: STRING v10.5 database and a confidence level of 0.7 were used. RESULTS: A highly interconnected network of 48 nodes was created, where insulin (INS) appears to be the major hub. INS upstream and downstream defects were analysed and revealed that only the kisspeptin- and glucagon-coding genes were upstream of INS. CONCLUSION: A metabolic dominance was inferred and discussed herein with its implications in puberty, obesity, infertility and cardiovascular function. This study, thus, may contribute to the resolution of a scientific conflict between the USA and EU definitions of the syndrome and/or provide a new P4 medicine approach.

Crosstalk between coagulation and complement activation promotes cardiac dysfunction in arrhythmogenic right ventricular cardiomyopathy.

Aims: We previously found that complement components are upregulated in the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), and inhibiting the complement receptor C5aR reduces disease severity in desmin knockout (Des-/- ) mice, a model for ARVC. Here, we examined the mechanism underlying complement activation in ARVC, revealing a potential new therapeutic target. Methods: First, immunostaining, RT-PCR and western blot were used to detect the expression levels of complement and coagulation factors. Second, we knocked out the central complement component C3 in Des-/- mice (ARVC model) by crossing Des-/- mice with C3-/- mice to explore whether complement system activation occurs independently of the conventional pathway. Then, we evaluated whether a targeted intervention to coagulation system is effective to reduce myocardium injury. Finally, the plasma sC5b9 level was assessed to investigate the role in predicting adverse cardiac events in the ARVC cohort. Results: The complement system is activated in the myocardium in ARVC. Autoantibodies against myocardial proteins provided a possible mechanism underlying. Moreover, we found increased levels of myocardial C5 and the serum C5a in Des-/-C3-/- mice compared to wild-type mice, indicating that C5 is activated independently from the conventional pathway, presumably via the coagulation system. Crosstalk between the complement and coagulation systems exacerbated the myocardial injury in ARVC mice, and this injury was reduced by using the thrombin inhibitor lepirudin. In addition, we found significantly elevated plasma levels of sC5b9 and thrombin in patients, and this increase was correlated with all-cause mortality. Conclusions: These results suggest that crosstalk between the coagulation and complement systems plays a pathogenic role in cardiac dysfunction in ARVC. Thus, understanding this crosstalk may have important clinical implications with respect to diagnosing and treating ARVC.

Vascular conditioning prevents adverse left ventricular remodelling after acute myocardial infarction: a randomised remote conditioning study.

AIMS: Remote ischemic conditioning (RIC) alleviates ischemia-reperfusion injury via several pathways, including micro-RNAs (miRs) expression and oxidative stress modulation. We investigated the effects of RIC on endothelial glycocalyx, arterial stiffness, LV remodelling, and the underlying mediators within the vasculature as a target for protection. METHODS AND RESULTS: We block-randomised 270 patients within 48 h of STEMI post-PCI to either one or two cycles of bilateral brachial cuff inflation, and a control group without RIC. We measured: (a) the perfusion boundary region (PBR) of the sublingual arterial microvessels to assess glycocalyx integrity; (b) the carotid-femoral pulse wave velocity (PWV); (c) miR-144,-150,-21,-208, nitrate-nitrite (NOx) and malondialdehyde (MDA) plasma levels at baseline (T0) and 40 min after RIC onset (T3); and (d) LV volumes at baseline and after one year. Compared to baseline, there was a greater PBR and PWV decrease, miR-144 and NOx levels increase (p < 0.05) at T3 following single- than double-cycle inflation (PBR:ΔT0-T3 = 0.249 ± 0.033 vs 0.126 ± 0.034 µm, p = 0.03 and PWV:0.4 ± 0.21 vs -1.02 ± 0.24 m/s, p = 0.03). Increased miR-150,-21,-208 (p < 0.05) and reduced MDA was observed after both protocols. Increased miR-144 was related to PWV reduction (r = 0.763, p < 0.001) after the first-cycle inflation in both protocols. After one year, single-cycle RIC was associated with LV end-systolic volume reduction (LVESV) > 15% (odds-ratio of 3.75, p = 0.029). MiR-144 and PWV changes post-RIC were interrelated and associated with LVESV reduction at follow-up (r = 0.40 and 0.37, p < 0.05), in the single-cycle RIC. CONCLUSION: RIC evokes "vascular conditioning" likely by upregulation of cardio-protective microRNAs, NOx production, and oxidative stress reduction, facilitating reverse LV remodelling. CLINICAL TRIAL REGISTRATION: http://www.clinicaltrials.gov . Unique identifier: NCT03984123.

The effect of Remote Ischemic Preconditioning (RIPC) on myocardial injury and inflammation in patients with severe aortic valve stenosis undergoing Transcatheter Aortic Valve Replacement (TAVΙ).

BACKGROUND: Remote ischemic preconditioning (RIPC) is being evaluated as a strategy to reduce cardiac injury and inflammation in patients undergoing diverse cardiac invasive and surgical procedures. However, it is unclear whether RIPC has protective effects in patients undergoing the transfemoral- transcatheter aortic valve implantation (TF-TAVΙ) procedure. METHODS: Between September 2013 and September 2015, 55 random consecutive patients were prospectively assigned to receive SHAM preconditioning (SHAM, 22 patients) or Remote Ischemic Preconditioning (RIPC) (4 cycles of 5 min intermittent leg ischemia and 5 min reperfusion, 33 patients) prior to TF-TAVI. The primary endpoint was to determine the serum levels of: hs-cTn-I (necrosis), CK-18 (apoptosis), and IL-1b (inflammation). Quantification was performed using commercially available ELISA kits. Patients were sampled 1-day pre TF-TAVΙ and 24-hours post TF-TAVΙ. Secondary endpoints included: total mortality, incidence of periprocedural clinical acute myocardial infarction (AMI), acute kidney injury (AKI), and stroke. RESULTS: 22 SHAM patients and 33 RIPC patients were finally analyzed. Our data revealed no significant difference in serum levels of hs-cTn-I and CK-18 among various groups. However, in the RIPC group, the increase in IL1b level was significantly lower for 24-h post TF-TAVΙ, (p < 0.01). There were no significant differences between groups in the secondary endpoints at the follow-up interval of one month. RIPC-related adverse events were not observed. CONCLUSIONS: Our data suggest that RIPC did not exhibit significant cardiac or kidney protective effects regarding necrosis and apoptosis in patients undergoing TF-TAVΙ. However, an important anti-inflammatory effect was detected in the RIPC group.

Tissue-specific relaxin-2 is differentially associated with the presence/size of an arterial aneurysm and the severity of atherosclerotic disease in humans.

Circulating or tissue-related biomarkers are of clinical value for risk stratification in patients with abdominal aortic aneurysms. Relaxin-2 (RL2) has been linked to the presence and size of arterial aneurysms, and to the extent of atherosclerosis in human subjects. Here, we assessed the expression levels of RL2 in aneurysmal (AA, n = 16) and atherosclerotic (ATH, n = 22) arteries, and established the correlation between RL2 levels and the presence/size of AA and the clinical severity of atherosclerosis. The expression levels of metalloproteinases (MMPs) and endothelial nitric oxide synthetase (eNOS) were also detected for correlations with different phenotypes of atherosclerosis and AA. Temporal artery biopsy specimens (n = 6) and abdominal aortic tissues harvested from accident victims during autopsy (n = 10) were used as controls. Quantitative tissue biomarker analysis revealed that tissue-specific RL2 was increased in patients with larger or symptomatic AA compared to subjects with atherosclerotic disease and healthy controls. In situ RL2 levels were proportional to the size and the severity of aneurysmatic disease, and were substantially elevated in patients with symptomatic aneurysm of any diameter or asymptomatic aneurysm of a diameter >350% of that of the normal artery. In contrast, tissue RL2 was inversely associated with the clinical severity of atherosclerotic lesions. Correlation between RL2 and MMP2 was different between ATH1 and ATH2, depending on atherosclerosis grade. Overall, tissue RL2 is differentially associated with discrete phenotypes of arterial disease and might exert multipotent biological effects on vascular wall integrity and remodeling in human subjects.

Levosimendan prevents doxorubicin-induced cardiotoxicity in time- and dose-dependent manner: implications for inotropy.

AIMS: Levosimendan (LEVO) a clinically-used inodilator, exerts multifaceted cardioprotective effects. Case-studies indicate protection against doxorubicin (DXR)-induced cardiotoxicity, but this effect remains obscure. We investigated the effect and mechanism of different regimens of levosimendan on sub-chronic and chronic doxorubicin cardiotoxicity. METHODS AND RESULTS: Based on preliminary in vivo experiments, rats serving as a sub-chronic model of doxorubicin-cardiotoxicity and were divided into: Control (N/S-0.9%), DXR (18 mg/kg-cumulative), DXR+LEVO (LEVO, 24 µg/kg-cumulative), and DXR+LEVO (acute) (LEVO, 24 µg/kg-bolus) for 14 days. Protein kinase-B (Akt), endothelial nitric oxide synthase (eNOS), and protein kinase-A and G (PKA/PKG) pathways emerged as contributors to the cardioprotection, converging onto phospholamban (PLN). To verify the contribution of PLN, phospholamban knockout (PLN-/-) mice were assigned to PLN-/-/Control (N/S-0.9%), PLN-/-/DXR (18 mg/kg), and PLN-/-/DXR+LEVO (ac) for 14 days. Furthermore, female breast cancer-bearing (BC) mice were divided into: Control (normal saline 0.9%, N/S 0.9%), DXR (18 mg/kg), LEVO, and DXR+LEVO (LEVO, 24 µg/kg-bolus) for 28 days. Echocardiography was performed in all protocols. To elucidate levosimendan's cardioprotective mechanism, primary cardiomyocytes were treated with doxorubicin or/and levosimendan and with N omega-nitro-L-arginine methyl ester (L-NAME), DT-2, and H-89 (eNOS, PKG, and PKA inhibitors, respectively); cardiomyocyte-toxicity was assessed. Single bolus administration of levosimendan abrogated DXR-induced cardiotoxicity and activated Akt/eNOS and cAMP-PKA/cGMP-PKG/PLN pathways but failed to exert cardioprotection in PLN-/- mice. Levosimendan's cardioprotection was also evident in the BC model. Finally, in vitro PKA inhibition abrogated levosimendan-mediated cardioprotection, indicating that its cardioprotection is cAMP-PKA dependent, while levosimendan preponderated over milrinone and dobutamine, by ameliorating calcium overload. CONCLUSION: Single dose levosimendan prevented doxorubicin cardiotoxicity through a cAMP-PKA-PLN pathway, highlighting the role of inotropy in doxorubicin cardiotoxicity.

Differential expression patterns of Toll Like Receptors and Interleukin-37 between calcific aortic and mitral valve cusps in humans.

BACKGROUND: Significant differences are mentioned in the progress of calcification between aortic and mitral valve. Evidence of inflammation in calcific aortic and mitral valve disease suggests that pathways of Toll Like Receptors (TLR) and Interleukin (IL)-37 expression may contribute to this process. We sought to investigate the role of TLR-mediated inflammatory response and IL-37 pathway expression on aortic and mitral valve calcification. MATERIAL AND METHODS: One-hundred twenty stenotic valve cusps/leaflets (60 aortic, 60 mitral) were excised during surgery and were collected for histological, immunohistochemistry and morphometric analysis at our department. After total RNA isolation from a second part of valve cusps/leaflets, cDNA synthesis and quantitative reverse transcription polymerase chain reaction (qRT-PCR) protocols were performed and relative mRNA levels of target genes were assessed. RESULTS: By histological analysis, the anti-inflammatory IL-37 levels were increased in mitral valve leaflets (MVL) compared to aortic valve cusps (AVCu) while all other biomarkers, including TLR, presented a reverse pattern with decreased levels as compared to AVCu. In terms of calcification biomarkers, only osteopontin differed between AVCu and MVL. mRNA analysis confirmed increased expression of IL-37 and decreased levels of TLR in MVL compared to AVCu. CONCLUSIONS: Stenotic cusps of aortic valves express lower IL-37 and increased TLRs levels than stenotic mitral valve leaflets, suggesting a differential pro-calcification and pro-inflammatory profile between the two valves. This may explain the higher incidence of calcification of AVCu than MVL and offer therapeutic considerations.