Pseudihypoxia in renal cell carcinoma

Циљ: Тумор реналних ћелија (RCC) је високо васкуларизовани тумор са израженом способношћу деобе ћелија захваљујући смањеној периферној концентрацији кисеоника. Цео систем хипоксија индуцибилних протеина представља значајан патогенетски механизам у настанку и промоцији тумора бубрега, као и настанку повећане отпорности на терапију са тирозин киназним инхибиторима (TKИ) и блокаторима васкуларног ендотелног фактора раста (VEGF) и његових рецептора. Главни циљеви рада су: 1) секвенцирање von Hippel-Lindau (VHL) гена и утицај на активност хипоксија активираних гена као што су хипоксија индуцубилни фактор (HIF-1α), који следствено активира еритропоетин (ЕРО) и VEGF и њихове рецепторе. 2) испитивање регулације VHL активности кисеоник зависним (пролил хидроксилазе-PHD) и независним путевима (“heat shock протеин-Hsp”). 3) поређење митоген активишуће протеин киназе (МАРК) и фосфатидилинозитол-3 киназе (PI-3), пролиферативних путева у туморском ткиву и здравом ткиву, као и активност „Janus kinazе” и “Signal Transducer and Activator of Transcription” (JAK2-STAT5) пута. 4) експресија гена и сигналних путева у ендотелијалним ћелијама гајених у нормалним и хипоскичним условима као модела развоја крвних судова у туморском ткиву. Методологија: У студију је укључено 50 пацијената који су били индиковани за хируршки захват тј. парцијалну или тоталну нефректомију. Локални етички комитет Клиничког центра Србије је одобрио студију. Пацијенти су били упознати са студијом, тако да се пре почетка операције узимала крв у цитрату за изолацију ДНК и након операције узимао се мали исечак туморског и здравог ткива за изолацију РНК, ДНК и протеина. Коришћен модел ендотелијалних ћелија које су гајене у нормалним и хипоксичним условима и касније изолована РНК и протеини за анализу...Objective: Renal cell carcinoma (RCC) is highly vascularized and proliferative tumor in relation to reduced oxygen tension, The entire system of hypoxia-inducible proteins represents a relevant pathogenetic mechanism in the initiation and promotion of renal tumors as well as development of enhanced therapy resistance to anti-angiogenic drugs and tyrosine kinase inhibitors. The aims of this study were: 1) to sequence von Hippel-Lindau (VHL) gene and to examine the influence of mutations in VHL gene on hypoxia activated genes, like hypoxia inducible factor 1 (HIF-1α) together with erythropoietin (ЕРО) and vascular endothelial growth factor (VEGF) and their receptors. 2) to estimate the regulation of VHL activity by oxygen dependent prolil hydroxylases (PHD) and independent heat shock protein (Hsp) pathway. 3) to compare two major proliferative pathways МАРК (mitogen activated protein kinase) and PI-3 (phosphatidylinositol 3-kinase) in tumor and healthy tissue, and activity of Janus kinazе and Signal Transducer and Activator of Transcription (JAK2-STAT5) pathway. 4) to identify activated genes and signaling pathways in endothelial cells under low and normal oxygen tension, as a model for oxygen regulation and proliferation of endothelial cells in tumor tissue. Methodology: In our study we analyzed 50 renal tumor and surrounding normal tissue samples of patients after radical nephrectomy, for DNA, RNA and protein analysis. Together with tissues, blood samples were collected for DNA isolation. This study was approved by the local comity of Clinical Center of Serbia. Primary endothelial cells and endothelial cell lines were cultured under low and normal oxygen tension and used for RNA and protein extraction. Results: With direct sequencing and multiplex ligation-dependent probe amplification (MLPA) methods of VHL gene, in tumors and surrounding healthy tissues, somatic mutations in VHL gene were present in 58% of all tumor samples

Stvaranje azot-monoksida izazvano bradikininom nije dovoljno za indukciju gama-globina

Introduction Hydroxycarbamide, used in therapy of hemoglobinopathies, enhances nitric oxide (NO) production both in primary human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cell line (TrHBMEC). Moreover, NO increases γ-globin and fetal hemoglobin levels in human erythroid progenitors. Objective In order to find out whether simple physiologic stimulation of NO production by components of hematopoietic microenvironment can increase γ-globin gene expression, the effects of NO-inducer bradykinin were examined in endothelial cells. Methods The study was performed in co-cultures of human erythroid progenitors, TrHBMEC and HUVECs by ozone-based chemiluminescent determination of NO and real-time quantitative RT-PCR. Results In accordance with previous reports, the endogenous factor bradykinin increased endothelial cell production of NO in a dose- and time-dependent manner (0.1-0.6 μM up to 30 minutes). This induction of NO in HUVECs and TrHBMEC by bradykinin was blocked by competitive inhibitors of NO synthase (NOS), demonstrating NOS-dependence. It has been shown that bradykinin significantly reduced endothelial NOS (eNOS) mRNA level and eNOS/s-actin ratio in HUVEC (by twofold). In addition, bradykinin failed to increase γ-globin mRNA expression in erythroid progenitors only, as well as in co-culture studies of erythroid progenitors with TrHBMEC and HUVEC after 24 hours of treatment. Furthermore, bradykinin did not induce γ/β globin ratio in erythroid progenitors in co-cultures with HUVEC. Conclusion Bradykinin mediated eNOS activation leads to short time and low NO production in endothelial cells, insufficient to induce γ-globin gene expression. These results emphasized the significance of elevated and extended NO production in augmentation of γ-globin gene expression.Uvod Hidroksikarbamid, koji se koristi u lečenju hemoglobinopatija, podstiče stvaranje azot-monoksida (NO) kako u primarnim ljudskim endotelnim ćelijama pupčane vene (HUVEC), tako i u izmenjenoj endotelnoj ćelijskoj liniji poreklom iz koštane srži (TrHBMEC). Štaviše, NO povećava stvaranje γ-globina i fetalnog hemoglobina u ljudskim progenitorima eritropoeze. Cilj rada Da bismo ustanovili da li jednostavna fiziološka stimulacija stvaranja NO od komponenti mikrosredine hematopoeze može povećati ekspresiju γ-globinskog gena, ispitivali smo efekte bradikinina, već poznatog stimulatora stvaranja NO. Metode rada Studija je izvedena u zajedničkim kulturama ljudskih progenitora eritropoeze sa TrHBMEC ili HUVEC i ispitivana hemiluminiscentnim merenjem NO posredstvom ozona, kao i primenom kvantitativnog RT-PCR na genskom nivou. Rezultati U skladu s prethodnim izveštajima, pokazali smo da endogeni faktor bradikinin povećava stvaranje NO u endotelnim ćelijama na dozno i vremenski zavisan način (0,1-0,6 μM do 30 minuta). Ovo stvaranje NO u HUVEC i TrHBMEC izazvano bradikininom blokirano je od strane konkurentskih inhibitora NO-sintaze (NOS), pokazujući NOS-zavisnost. Utvrdili smo da bradikinin značajno smanjuje stvaranje iRNK endotelne forme NOS (eNOS), kao i odnos eNOS i β-aktina u HUVEC (dvostruko manje). Pored toga, bradikinin ne povećava ekspresiju iRNK γ-globinskog gena ni u zasebnim progenitorima eritropoeze, niti u zajedničkim kulturama progenitora eritropoeze sa TrHBMEC ili HUVEC posle 24 sata tretmana. Bradikinin ne menja ni odnos γ i β globina u zajedničkim kulturama progenitora eritropoeze sa HUVEC. Zaključak Aktivacija eNO_ izazvana bradikininom dovodi do kratkog i malog povećanja NO u endotelnim ćelijama, što je nedovoljno da podstakne ekspresiju gena za γ-globin. Ovi rezultati naglašavaju važnost povećanog i produženog stvaranja NO radi stimulacije ekspresije γ-globina

Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells

Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 pM of NO donor diethylenetriamine NONOate (DETANO) for 24 h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 uM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR Furthermore, DETANO stimulated Ala anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO response in endothelial cells, particularly at low oxygen tensions

Proinflammatory Cytokine IL-6 and JAK-STAT Signaling Pathway in Myeloproliferative Neoplasms

The recent JAK1/2 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be more beneficial than targeting gene mutants. We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway related genes in circulating CD34(+) cells of MPNs. Regarding laboratory data, leukocytosis has been observed in polycythemia vera (PV) and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2V617F allele burden in essential thrombocythemia (ET) and PMF. 261 significantly changed genes have been detected in PV, 82 in ET, and 94 genes in PMF. The following JAK-STAT signaling pathway related genes had augmented expression in CD34(+) cells of MPNs: CCND3 and IL23A regardless of JAK2V617F allele burden; CSF3R, IL6ST, and STAT1/2 in ET and PV with JAK2V617F mutation; and AKT2, IFNGR2, PIM1, PTPN11, and STAT3 only in PV. STAT5A gene expression was generally reduced in MPNs. IL-6 cytokine levels were increased in plasma, as well as IL-6 protein levels in bone marrow stroma of MPNs, dependent on JAK2V617F mutation presence in ET and PMF patients. Therefore, the JAK2V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs

Low oxygen tension primes aortic endothelial cells to the reparative effect of tissue-protective cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared to 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis

Effects of the polyphenol resveratrol on contractility of human term pregnant myometrium

The ideal agent for prevention and treatment of uterine abnormal contractility has not been found. The polyphenol resveratrol possesses a wide spectrum of pharmacologic properties, but its influence on the contractility of human myometrium is not defined. The present study evaluated the effect of resveratrol on the oxytocin-induced contractions of human term pregnant myometrium in vitro and the contribution of different K+ channels to resveratrol action. Resveratrol induced a concentration-dependent relaxation of myometrium contractions (pD(2) value and maximal responses were 4.52 and 82.25%, respectively). Glibenclamide, a selective blocker of ATP-sensitive (K-ATP), iberiotoxin, a selective blockers of big-calcium sensitive (BKCa) and 4-aminopiridine, a non-selective blocker of voltage-sensitive (Kv) channels induced a significant shift to the right of the concentration-response curves of resveratrol. Inhibition achieved by 0.1 mM resveratrol was insensitive to all K+ channel blockers. A K+ channel opener, pinacidil, inhibited oxytocin-induced contractions of pregnant myometrium with comparable potency and efficacy to resveratrol (pD(2) values and maximal relaxation were 4.52 and 83.67%, respectively). Based on K+ channel opener/blocker affinities, it appears that the inhibitory response of resveratrol involves different myometrial K+ channels. When applied in high concentrations, resveratrol has an additional K+-channel-independent mechanism(s) of action. Furthermore, immunohistochemistry staining and western blot analyses detected the presence and distribution of K-ATP, BKCa and Kv channel proteins in pregnant myometrium

Stimulated stromal cells induce gamma-globin gene expression in erythroid cells via nitric oxide production

Objective. We have previously shown that nitric oxide (NO) is involved in the hydroxyurea-induced increase of gamma-globin gene expression in cultured human erythroid progenitor cells and that hydroxyurea increases NO production in endothelial cells via endothelial NO synthase (NOS). We have now expanded those studies to demonstrate that stimulation of gamma-globin gene expression is also mediated by NOS induction in stromal cells within the bone marrow microenvironment. Materials and Methods. Using NO analyzer, we measured NO production in endothelial and macrophage cell cultures. In coculture studies of erythroid and stromal cells, we measured globin gene expression during stimulation by NO induers. Results. Hydroxyurea (30 - 100 mu M) induced NOS-dependent production of NO in human macrophages (up to 1.2 mu M). Coculture studies of human macrophages with erythroid progenitor cells also resulted in induction of gamma-globin mRNA expression (up to threefold) in the presence of hydroxyurea. NOS-dependent stimulation of NO by lipopolysaccharide (up to 0.6 mu M) has been observed in human macrophages. We found that lipopolysaccharide and interferon-gamma together increased gamma-globin gene expression (up to twofold) in human macrophage/erythroid cell cocultures. Coculture of human bone marrow endothelial cells with erythroid progenitor cells also induced gamma-globin mRNA expression (2.4-fold) in the presence of hydroxyurea (40 mu M). Conclusion. These results demonstrate an arrangement by which NO and fetal hemoglobin inducers may stimulate globin genes in erythroid cells via the common paracrine effect of bone marrow stromal cells

Dedicated bifurcation analysis: basic principles

Over the last several years significant interest has arisen in bifurcation stenting, in particular stimulated by the European Bifurcation Club. Traditional straight vessel analysis by QCA does not satisfy the requirements for such complex morphologies anymore. To come up with practical solutions, we have developed two models, a Y-shape and a T-shape model, suitable for bifurcation QCA analysis depending on the specific anatomy of the coronary bifurcation. The principles of these models are described in this paper, as well as the results of validation studies carried out on clinical materials. It can be concluded that the accuracy, precision and applicability of these new bifurcation analyses are conform the general guidelines that have been set many years ago for conventional QCA-analyses