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Purpose: Endothelial derivatives of human pluripotent stem cells hold out hope for therapeutic angiogenesis. Our aim was to investigate the effect of differentiation protocols on the development of arterial and venous endothelial cells and to study the arterial and venous gene expression pattern after in vivo engraftment of endothelial cells.Methods: H7 human embryonic stem cells (hESC, from Wicell) were differentiated via embryoid body (EB) formation method in normoxygenic (20%) and hypoxic (5%) conditions as well as via monolayer method in the presence of vascular-endothelial growth factor (VEGF, 1ng/ml). Human induced pluripotent stem cells (hIPSC, from ReproCell) were differentiated under normoxygenic condition via EB formation. CD31-positive endothelial cells (EC) were sorted by FACS. For engineering 3D constructs, human aortic wall samples were decellularised with detergent solution (2% sodium dodecyl sulfate) and hESC-EC and hIPSC-EC were seeded onto this extracellular matrix. Human ESC-EC and hIPSC-EC were transplanted into three months old athymic nude rats using Matrigel as an extracellular matrix carrier.Results: The mRNA levels of angiopoietin2 showed an increase in hESC-EC when differentiated with EB method (EB normoxia 353.17±86.29; EB hypoxia 323.89±86.63, p<0.001, n=3, mRNA levels normalized to those in undifferentiated hESC). As shown by immunocytochemistry, differentiated hESC-EC and hIPSC-EC were stained positive for anti-CD31, von Willebrand factor and ve-cadherin; cells formed capillary-like tubules on Matrigel and took up acetylated-LDL. Quantitative PCR showed abundant expressions of both arterial (EphrinB2, Notch1-2) and venous (EphB4) endothelial markers; however, lymphatic endothelial cells were not detectable. As assessed by proteome profiling, a wide range of angiogenesis-related proteins were detected in both in hESC-EC and hIPSC-EC. Human ESC-EC and hIPSC-EC seeded onto decellularised human extracellular matrix remained viable as shown by calcein AM staining. Cells remained viable also upon in vivo engraftment; marked increase was found in mRNA levels of all arterial and venous marker genes in re-isolated cells (EphrinB2, EphB4, Notch 1-2, CD31, n=3). Conclusions: EB-based differentiation protocol of endothelial cells from human pluripotent stem cells has the highest angiogenic potency in vitro. After in vivo conditioning, expression of endothelial markers increased, suggesting the functional role of engrafted endothelial cells and possibly supporting future therapeutic purposes with specific angiogenic cells. [ABSTRACT FROM PUBLISHER]

Purpose: Endothelial cells have an essential role in the maintenance of cardiac function. Cardiac endothelial cells in response of different cardiovascular risk factors become activated and this activation is often followed by endothelial dysfunction. The change of endothelial phenotype may influence interactions with surrounding cardiac fibroblasts, myocytes, pericytes.The aim of our work is to produce in vitro culture system with high cell purity to investigate endothelial cells in different heart diseases. Poor quality of these tissue samples indicates the optimalization of isolation and culture conditions for both cardiac microvascular and endocardial endothelial cells (CMVE and EE cells).Methods: Human heart samples were obtained from valve surgery or explanted hearts during transplantation. CMVE cells were isolated from left ventricle with collagenase and trypsin digestion. EE cells were digested with collagenase from mitral valve. After separation the pellets were seeded to culture dishes and grown in a medium containing endothelial cell growth supplement. These basic methods would be changed as follows. The time of enzymatic digestion was changed. The collected cell suspensions were preincubated to allow fibroblasts to attach to the bottom of culture dish. The decanting medium was centrifuged repeatedly and collected cells were cultured in specific endothelial medium completed with sera from different origins. CMVE cells were treated with puromycin to eliminate pericytes. Cells were seeded on different artificial extracellular surface components (collagen, laminin, gelatin, fibronectin). The CMVE and EE cells were characterized by uptake of acetylated LDL and immunostaining of vonWillebrand factor, VE-cadherin and CD31. Fibroblasts were identified by vimentin staining.Results: Primary cells isolated with basic methods exhibited different cell morfologies, they were multilayered and only the 4% and 10% of the cells were endothelial cells, CMVE and EE respectively, separated by flourescent-activated cell sorting (FACS). The attachment of fibroblasts and the puromycin treatment were effective in the primary cell cultures. Numerous cell colonies were observed within 2-3 days on gelatin coated surface and confluence was achieved within 10-14 days.Conclusion: With use of these culture conditions the endothelial cell rate is increased in primery cultures and further separation by FACS or immunomagnetic methods will be more effective. These optimalized cell culture systems provide possibility to investigate endothelial functions in different heart diseases. [ABSTRACT FROM PUBLISHER]

Purpose: Vascular derivatives of human embryonic stem cells (hESC) are being developed as sources of tissue-specific cells for organ regeneration. However, identity of developmental pathways that modulate the specification of endothelial cells is not known yet.Methods: We studied PI3K-FOXO1A pathways during differentiation of H7 hESC towards endothelial lineage and on proliferation, maturation and cell death of hESC-derived endothelial cells (hESC-EC). We differentiated hESC towards endothelial lineage and used FACS to isolate CD31+ cells at day 13 after initiation of differentiation.Results: During differentiation of hESC, expression of FOXO1A transcription factor was linked to the expression of a cluster of angiogenesis- and vascular remodelling-related genes. PI3K inhibitor LY294002 (10 μM) activated FOXO1A as shown by real-time PCR and nuclear translocation assay, and induced formation of CD31+ hESC-EC. In contrast, differentiating hESC with silenced FOXO1A by siRNA showed lower mRNA levels of CD31 and angiopoietin2. Similarly, overexpression of FOXO1A-eGFP construct in hESC-EC resulted in an increased angiopoietin2 expression (1.5-fold, p<0.5, n=3) as well as higher percentage of Topro3-positive necrotic cells (p<0.001). LY294002 decreased proliferative activity of purified hESC-EC, whilst FOXO1A siRNA increased their proliferation. LY294002 inhibited migration and tube formation of hESC-EC; in contrast, FOXO1A siRNA increased in vitro tube formation activity of hESC-EC. Using a small-animal PET/MRI system along with gallium-labelled NOTA-based conjugates for in vivo multimodality imaging showed an active angiogenic activity of hESC-EC in athymic nude rats. After in vivo conditioning of cells for three weeks, cells retain their low FOXO1A expression levels.Conclusions: PI3K/FOXO1A pathway is important for function and survival of hESC-EC as well as in the regulation of endothelial cell fate. Understanding these properties of hESC-EC may help in future applications for treatment of injured organs. [ABSTRACT FROM PUBLISHER]

a-Methylacyl coenzyme A racemase (AMACR) is traditionally considered to be a marker of papillary renal cell carcinoma. However, AMACR expression can be seen in other renal tumors. The aim of this study was to investigate AMACR immunoreactivity within the spectrum of clear cell renal cell neoplasms. Fifty-three clear cell renal epithelial tumors were used in assembling the following four cohorts: low grade (LG) clear cell renal cell carcinoma (CCRCC), high grade (HG) CCRCC, CCRCC with cystic changes, and multilocular cystic renal neoplasm of low malignant potential (MCRNLMP). Representative blocks were stained for AMACR, using two different clones (SP52 and OV-TL12/30). There were at least some AMACR immunoreactivity in 77.8 % and 68.9 % of CCRCCs (using SP52 and OV-TL12/30 clone, respectively). Moderate to strong positivity, or positivity in more than one third of the tumor (even weak in intensity) was detected in 46.7 % of CCRCCs using SP52 and in 48.9 % of CCRCC using OV-TL12/30 clone. The highest AMACR reactivity was observed in HG CCRCC (60 % by SP52 and 66.7 % by OV-TL12/30). Strong and diffuse AMACR positivity was detected in 8.9 % of all CCRCCs. AMACR immunoreactivity in MCRNLMP was 37.5 % (SP52 clone) and 25 % (OV-TL12/30 clone). We demonstrated relatively high expression rate of AMACR in CCRCC, while very variable in intensity and distribution. This finding may have diagnostic implications especially in limited samples (i.e., core biopsies), as AMACR positivity does not exclude the diagnosis of CCRCC., Competing Interests: Declaration of competing interest The authors declare no financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ondrej Fiala received honoraria from Novartis, Janssen, Merck and Pfizer for consultations and lectures unrelated to this project., (Copyright © 2024 Elsevier Inc. All rights reserved.)

A syndromic association between a subset of testicular/paratesticular neoplasms is well established. Such examples include Carney complex and large cell calcifying Sertoli cell tumor, Peutz-Jeghers syndrome and intratubular large cell hyalinizing Sertoli cell neoplasia, and VHL syndrome and clear cell papillary cystadenoma of the epididymis.However, recent studies proposed potential novel links between some testicular and paratesticular neoplasms with certain tumor syndromes. While more studies are still needed to solidify these associations, recent research suggests that a subset of Leydig cell tumors may arise in patients with hereditary leiomyomatosis and renal cell carcinoma syndrome or that some seminomas may occur in Lynch syndrome patients. Additionally, an association between testicular sex cord stromal tumors and paratesticular sarcomas with Familial adenomatous polyposis syndrome and DICER1 syndrome, respectively, has been proposed as well. This review provides a comprehensive overview of the intricate relationship between familial syndromes and associated testicular and paratesticular tumors, shedding light on their clinicopathological and molecular characteristics., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Upper tract urothelial carcinoma (UTUC) is the third most common malignancy associated with Lynch syndrome (LS). The current European urology guidelines recommend screening for LS in patients with UTUC up to the age of 60 years. In this study, we examined a cohort of patients with UTUC for potential association with LS in order to establish the sensitivity of current guidelines in detecting LS. A total of 180 patients with confirmed diagnosis of UTUC were enrolled in the study during a 12-year period (2010-2022). Loss of DNA-mismatch repair proteins (MMRp) expression was identified in 15/180 patients (8.3%). Germline analysis was eventually performed in 8 patients confirming LS in 5 patients (2.8%), including 4 germline mutations in MSH6 and 1 germline mutation in MSH2. LS-related UTUC included 3 females and 2 males, with a mean age of 66.2 years (median 71 years, range 46-75 years). Four of five LS patients (all with MSH6 mutation) were older than 65 years (mean age 71.3, median 72 years). Our findings indicate that LS-associated UTUCs can occur in patients with LS older than 60 years. In contrast to previous studies which used mainly highly pre-selected populations with already diagnosed LS, the most frequent mutation in our cohort involved MSH6 gene. All MSH6 mutation carriers were > 65 years, and UTUC was the first LS manifestation in 2/4 patients. Using current screening guidelines, a significant proportion of patients with LS-associated UTUC may be missed. We suggest universal immunohistochemical MMRp screening for all UTUCs, regardless of age and clinical history., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The significance of cardiology screening of referees is not well established. Cardiovascular risk factors and diseases were examined in asymptomatic Hungarian elite handball referees undergoing extended screening: personal/family history, physical examination, 12-lead ECG, laboratory tests, body-composition analysis, echocardiography, and cardiopulmonary exercise testing. Holter-ECG (n = 8), blood pressure monitorization (n = 10), cardiac magnetic resonance imaging (CMR; n = 27) and computer tomography (CCT; n = 4) were also carried out if needed. We examined 100 referees (age: 29.6±7.9years, male: 64, training: 4.3±2.0 hours/week), cardiovascular risk factors were: positive medical history: 24%, overweight: 10%, obesity: 3%, dyslipidaemia: 41%. Elevated resting blood pressure was measured in 38%. Stress-ECG was positive due to ECG-changes in 16%, due to elevated exercise blood pressure in 8%. Echocardiography and/or CMR identified abnormalities in 19%. A significant number of premature ventricular contractions was found on the Holter-ECG in two cases. The CCT showed myocardial bridge or coronary plaques in one-one case. We recommended lifestyle changes in 58%, new/modified antihypertensive or lipid-lowering therapy in 5%, iron-supplementation in 22%. By our results, a high percentage of elite Hungarian handball referees had cardiovascular risk factors or diseases, which, combined with physical and psychological stress, could increase the possibility of cardiovascular events. Our study draws attention to the importance of cardiac screening in elite handball referees., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Relaxin-1 (RLN1) has emerged as a possible therapeutic target in myocardial fibrosis due to its anti-fibrotic effects. Previous randomized clinical trials investigated therapeutic role of exogenous relaxin in patients with acute-on-chronic heart failure (HF) and failed to meet clinical endpoints. Here, we aimed to assess endogenous, circulating RLN1 levels in patients with heart failure with reduced ejection fraction (HFrEF) of ischemic origin. Furthermore, we analyzed relation of RLN1 and left ventricular diastolic function, left and right ventricular fibrosis, and invasive hemodynamic measurements. Unique feature of our study is the availability of ex vivo human myocardial tissue. Methods: Human myocardial samples were available from the Transplantation Biobank of the Heart and Vascular Center at Semmelweis University after local ethical approval and informed consent of all participants ( n = 47). Tissue was collected immediately after heart explantations; peripheral blood was collected before induction of anesthesia. Myocardial sections were stained for Masson's trichrome and Picrosirius red staining to quantify fibrosis. Medical records were analyzed (ECG, anthropometry, blood tests, medication, echocardiography, and invasive hemodynamic measurements). Results: Average RLN1 levels in HFrEF population were significantly higher than measured in age and gender matched healthy control human subjects (702 ± 283 pg/ml in HFrEF vs. 44 ± 27 pg/ml in control n = 47). We found a moderate inverse correlation between RLN1 levels and degree of myocardial fibrosis in both ventricles ( r = -0.357, p = 0.014 in the right ventricle vs. r = -0.321, p = 0.028 in the left ventricle with Masson's trichrome staining). Parallel, a moderate positive correlation was found in left ventricular diastolic function (echocardiography, E/A wave values) and RLN1 levels ( r = 0.456, p = 0.003); a negative correlation with RLN1 levels and left ventricular end-systolic diameter ( r = -0.373, p = 0.023), and diastolic pulmonary artery pressure ( r = -0.894, p < 0.001). RLN1 levels showed moderate correlation with RLN2 levels ( r = 0.453, p = 0.0003). Conclusion: Increased RLN1 levels were accompanied by lower myocardial fibrosis rate, which is a novel finding in our patient population with coronary artery disease and HFrEF. RLN1 can have a biomarker role in ventricular fibrosis; furthermore, it may influence hemodynamic and vasomotor activity via neurohormonal mechanisms of action. Given these valuable findings, RLN1 may be targeted in anti-fibrotic therapeutics and in perioperative care of heart transplantation.

Background: The archetypal DNA methyltransferase inhibitors, 5-azacytidine (AZA) and 5-aza-2'-deoxycytidine (DAC) are potent antineoplastic agents used in the treatment of mainly, blood malignancies. However, the administration of these drugs is confounded by their hydrolytic lability which decreases plasma circulation time. Here, we describe a new biodegradable, polyanhydride formulation for drug delivery that circumvents this drawback., Methods: Injectable/implantable polymeric microbeads containing dispersed microcrystals of hydrophilic AZA or DAC packed in a dry environment are protected from hydrolysis, until the hydrolytic zone reaches the core. Diclofenac is embedded into the formulation to decrease any local inflammation. The efficacy of the formulations was confirmed by monitoring the induced demethylation, and cytostatic/cytotoxic effects of continuous drug release from the time-course dissolution of the microbeads, using an in vitro developed cell based reporter system., Results: Poly(sebaccic acid-co-1,4-cyclohexanedicarboxylic acid) containing 30 wt. % drug showed zero-order release (R(2) = 0.984 for linear regression), and release rate of 10.0 %/h within the first 5 h, and subsequent slower release of the remaining drug, thus maintaining the level of drugs in the outer environment considerably longer than the typical plasma half-life of free azanucleosides. At lower concentrations, the differences between powder drug formulations and microbeads were very low or negligible, however, at higher concentrations, we discovered equivalent or increasing effects of the drugs loaded in microbeads., Conclusions: The study provides evidence that microbead formulations of the hydrolytically labile azanucleoside drugs could prevent their chemical decomposition in aqueous solution, and effectively increase plasma circulation time.

Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage.

The anti-apoptotic gene replacements could be an option in preventing hypoxia-induced neuronal loss. In this paper we tested the effect of anti-apoptosis (bcl-2 and bcl-XL) gene transfer on cell plasticity. Nestin, synapsin-1 and c-fos genes and proteins expression were measured in PC12 cells in normal condition, and after hypoxia/re-oxygenation. Gene delivery results a significant increase in both bcl-2 and bcl-XL gene expression. Hypoxia (1h)/re-oxygenation (24h) have a detrimental effect upon cultured cells by increasing the pro-apoptotic, bax gene and protein expression. Bcl-2 or bcl-XL gene delivery resulted in a significant increase in and the cellular levels of the corresponding mRNAs and proteins. Bcl-2 gene augmented the nestin gene and protein expression which has been compromised previously by the hypoxic event. Similarly c-fos mRNA and protein expression decreased significantly after hypoxia, while the anti-apoptotic gene treatment normalized c-fos expression. Synapsin-1 gene or protein expression remained about on the same level under normoxic conditions or following hypoxia after gene treatment. We can conclude that anti-apoptotic gene transfers activate neuronal plasticity proteins nestin and c-fos. This link on anti-apoptotic proteins and cell plasticity is a new finding.

Context: Many patients with chronic angina experience anginal episodes despite revascularization and antianginal medications. In a previous trial, antianginal monotherapy with ranolazine, a drug believed to partially inhibit fatty acid oxidation, increased treadmill exercise performance; however, its long-term efficacy and safety have not been studied in combination with beta-blockers or calcium antagonists in a large patient population with severe chronic angina., Objectives: To determine whether, at trough levels, ranolazine improves the total exercise time of patients who have symptoms of chronic angina and who experience angina and ischemia at low workloads despite taking standard doses of atenolol, amlodipine, or diltiazem and to determine times to angina onset and to electrocardiographic evidence of myocardial ischemia, effect on angina attacks and nitroglycerin use, and effect on long-term survival in an open-label observational study extension., Design, Setting, and Patients: A randomized, 3-group parallel, double-blind, placebo-controlled trial of 823 eligible adults with symptomatic chronic angina who were randomly assigned to receive placebo or 1 of 2 doses of ranolazine. Patients treated at the 118 participating ambulatory outpatient settings in several countries were enrolled in the Combination Assessment of Ranolazine In Stable Angina (CARISA) trial from July 1999 to August 2001 and followed up through October 31, 2002., Intervention: Patients received twice-daily placebo or 750 mg or 1000 mg of ranolazine. Treadmill exercise 12 hours (trough) and 4 hours (peak) after dosing was assessed after 2, 6 (trough only), and 12 weeks of treatment., Main Outcome Measures: Change in exercise duration, time to onset of angina, time to onset of ischemia, nitroglycerin use, and number of angina attacks., Results: Trough exercise duration increased by 115.6 seconds from baseline in both ranolazine groups (pooled) vs 91.7 seconds in the placebo group (P =.01). The times to angina and to electrocardiographic ischemia also increased in the ranolazine groups, at peak more than at trough. The increases did not depend on changes in blood pressure, heart rate, or background antianginal therapy and persisted throughout 12 weeks. Ranolazine reduced angina attacks and nitroglycerin use by about 1 per week vs placebo (P<.02). Survival of 750 patients taking ranolazine during the CARISA trial or its associated long-term open-label study was 98.4% in the first year and 95.9% in the second year., Conclusion: Twice-daily doses of ranolazine increased exercise capacity and provided additional antianginal relief to symptomatic patients with severe chronic angina taking standard doses of atenolol, amlodipine, or diltiazem, without evident adverse, long-term survival consequences over 1 to 2 years of therapy.