Analiza ekspresije faktora inflamacije S100A4, S100A8/9, S100A12 u mijeloproliferativnim neoplazmama: veza sa prisustvom mutacije u JAK2 genu

Myeloproliferative neoplasms (MPNs) belong to the group of malignant diseases of the blood, characterized by a clonal expansion of neoplastically transformed hematopoietic progenitors of the myeloid lineage in the bone marrow. MPN in the narrow sense includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In patients with MPN, the underlying disorder is the JAK2V617F mutation, which is a driving mutation in more than 95% of patients with PV and 50-60% of patients with ET and PMF. The JAK2V617F mutation leads to constitutive activation of JAK2 kinase and cytokine-independent cell growth. As a consequence, downstream signaling pathways (JAK2 / STAT3, PI3K-AKT, MAPK), which are involved in myeloproliferation, are activated. The presence of the JAK2V617F mutation in MPN in hematopoietic cells alters the activity of inflammatory signaling pathways, altering the production of reactive oxygen species in neutrophils, altering serum inflammatory cytokine levels, and altering reactivity to these cytokines. The main feature of chronic inflammation is the high level of circulating cytokines and chemokines, as well as the accumulation of reactive oxygen species, which leads to genetic instability and may be suitable for the formation and progression of neoplasms. In patients with MPN, a significant increase in many inflammatory mediators has been observed, including a group of calcium-binding proteins, called S100 proteins. Due to their tumorigenic and pro-inflammatory activity, the S100 family of proteins that potentially have the greatest effect on the formation and development of myeloproliferative neoplasms are S100A4, S100A8, S100A9 and S100A12, as several studies have shown their elevated levels in MPN. The aim of this dissertation was to investigate the gene expression and levels of S100A4, S100A8, S100A9 and S100A12 factors in MPN patients, study the effect of pro-inflammatory cytokine IL-6, anti-inflammatory cytokine IL-10, as well as JAK inhibitors on the levels of these proteins on human HEL 92.1.7 model cell line with a JAK2 mutation, as well as an examination of the signaling mechanisms by which IL-6 and IL-10 exert their effects on peripheral blood cells of patients with MPN and the human HEL 92.1.7 model. cell line. The results of this study showed that inflammation factors show an increase in levels, which is dependent on the presence of the JAK2V617F mutation in individual proteins. This increase in the level of inflammatory factors indicates that patients with MPN have chronic inflammation. Treatment with hydroxyurea and a combination of hydroxyurea and a specific JAK2 inhibitor decreases the levels of inflammatory factor in the HEL 92.1.7 cell line, indicating that the S100 proteins examined are expressed in a JAK2-dependent manner. The pro-inflammatory cytokine IL-6 decreased S100 protein levels in mononuclear cells of patients with MPN, and this decrease in MPN was mediated by NF-κB and PI3K signaling pathways. The anti-inflammatory cytokine IL-10 also reduced S100 protein levels, especially S100A8 and S100A9 in patients with ET, but mediated by the PI3K-AKT signaling pathway. IL-6 / IL-10-mediated regulation of S100 protein levels by MPNs is dominated by NF-κB and PI3K signaling pathways, whereas activation of the JAK-STAT signaling pathway is constitutive.Mijeloproliferativne neoplazme (MPN) spadaju u grupu malignih oboljenja krvi, za koje je karakteristična klonalna ekspanzija neoplastično transformisanih hematopoetskih progenitora mijeloidne loze u kostnoj srži. U MPN u užem smislu spadaju policitemija vera (PV), esencijalna trombocitemija (ET) i primarna mijelofibroza (PMF). Kod pacijenata sa MPN osnovni poremećaj predstavlja mutacija JAK2V617F, koja je pokretačka mutacija u više od 95% pacijenata sa PV i 50-60% pacijenata sa ET i PMF. JAK2V617F mutacija vodi konstitutivnoj aktivaciji JAK2 kinaze i rastu ćelija nezavisnom od citokina. Kao posledica, aktivirani su nishodni signalni putevi (JAK2/STAT3, PI3K-AKT, MAPK), koji su uključeni u mijeloproliferaciju. Prisustvo JAK2V617F mutacije kod MPN u hematopoetskim ćelijama menja aktivnost signalnih puteva značajnih za inflamaciju, pri čemu dolazi do promene nastajanja reaktivnih vrsta kiseonika kod neutrofila, promene nivoa inflamatornih citokina u serumu i izmenjene reaktivnosti na ove citokine. Glavna odlika hronične inflamacije jeste visok nivo citokina i hemokina u cirkulaciji, kao i nagomilavanje reaktivnih kiseoničnih vrsta, što dovodi do genetičke nestabilnosti i može biti pogodno za nastanak i progresiju neoplazmi. Kod pacijenata sa MPN uočeno je značajno povećanje brojnih medijatora inflamacije, među kojima je i grupa kalcijum vezujućih proteina, pod nazivom S100 proteini. Zbog svog tumorigenog i proinflamatornog delovanja, proteini S100 familije koji potencijalno imaju najveći efekat na nastanak i razvoj mijeloproliferativnih neoplazmi su S100A4, S100A8, S100A9 i S100A12, jer je u nekoliko studija pokazan njihov povišen nivo kod MPN. Cilj istraživanja ove disertacije bilo je ispitivanje genske ekspresije i nivoa S100A4, S100A8, S100A9 i S100A12 faktora kod MPN pacijenata, ispitivanje uticaja proinflamatornog citokina IL-6, antiinflamatornog citokina IL-10, kao i JAK inhibitora na nivoe ovih proteina na modelu humane HEL 92.1.7 ćelijske linije sa JAK2 mutacijom, kao i ispitivanje signalnih mehanizama putem kojih IL-6 i IL-10 ostvaruju svoje efekte na ćelije periferne krvi pacijenata sa MPN i modelu humane HEL 92.1.7. ćelijske linije. Rezultati ove studije su pokazali da faktori inflamacije pokazuju povećanje nivoa, koje zavisi od prisustva JAK2V617F mutacije kod pojedinačnih proteina. Navedeno povećanje nivoa faktora inflamacije ukazuje da kod obolelih od MPN postoji hronična inflamacija. Tretman hidroksiureom i kombinacijom hidroksiuree i specifičnog JAK2 inhibitora smanjuje nivoe faktora inflamacije u HEL 92.1.7 ćelijskoj liniji, što ukazuje da se ispitivani S100 proteini eksprimiraju na JAK2-zavistan način. Proinflamatorni citokin IL-6 smanjivao je nivoe S100 proteina u mononuklearima pacijenata sa MPN, a ovo smanjenje kod MPN posredovano je NF-κB i PI3K signalnim putevima. Antiinflamatorni citokin IL-10 takođe je smanjivao nivoe S100 proteina, pogotovo S100A8 i S100A9 kod pacijenata sa ET, ali posredovano PI3K-AKT signalnim putem. U IL-6 / IL-10 posredovanoj regulaciji nivoa S100 proteina kod MPN dominiraju NF-κB i PI3K signalni putevi, dok je aktivacija JAK-STAT signalnog puta konstitutivna

Expression analysis of inflamation factors S100A4, S100A8/9, S100A12 in myeloproliferative neoplasms: a connection with the presence of mutation in the JAK2 gene

Мијелопролиферативне неоплазме (МПН) спадају у групу малигних обољења крви, за које је карактеристична клонална експанзија неопластично трансформисаних хематопоетских прогенитора мијелоидне лозе у костној сржи. У МПН у ужем смислу спадају полицитемија вера (ПВ), есенцијална тромбоцитемија (ЕТ) и примарна мијелофиброза (ПМФ). Код пацијената са МПН основни поремећај представља мутација JAK2V617F, која је покретачка мутација у више од 95% пацијената са ПВ и 50-60% пацијената са ЕТ и ПМФ. JAK2V617F мутација води конститутивној активацији ЈАК2 киназе и расту ћелија независном од цитокина. Као последица, активирани су нисходни сигнални путеви (JAK2/STAT3, PI3K-АКТ, MAPK), који су укључени у мијелопролиферацију. Присуство JAK2V617F мутације код МПН у хематопоетским ћелијама мења активност сигналних путева значајних за инфламацију, при чему долази до промене настајања реактивних врста кисеоника код неутрофила, промене нивоа инфламаторних цитокина у серуму и измењене реактивности на ове цитокине. Главна одлика хроничне инфламације јесте висок ниво цитокина и хемокина у циркулацији, као и нагомилавање реактивних кисеоничних врста, што доводи до генетичке нестабилности и може бити погодно за настанак и прогресију неоплазми. Код пацијената са МПН уочено је значајно повећање бројних медијатора инфламације, међу којима је и група калцијум везујућих протеина, под називом S100 протеини. Због свог туморигеног и проинфламаторног деловања, протеини S100 фамилије који потенцијално имају највећи ефекат на настанак и развој мијелопролиферативних неоплазми су S100А4, S100А8, S100А9 и S100А12, јер је у неколико студија показан њихов повишен ниво код МПН. Циљ истраживања ове дисертације било је испитивање генске експресије и нивоа S100A4, S100A8, S100A9 и S100A12 фактора код МПН пацијената, испитивање утицаја проинфламаторног цитокина IL-6, антиинфламаторног цитокина IL-10, као и JAK инхибитора на нивое ових протеина на моделу хумане HEL 92.1.7 ћелијске линије са ЈАК2 мутацијом, као и испитивање сигналних механизама путем којих IL-6 и IL-10 остварују своје ефекте на ћелије периферне крви пацијената са МПН и моделу хумане HEL 92.1.7. ћелијске линије. Резултати ове студије су показали да фактори инфламације показују повећање нивоа, које зависи од присуства JAK2V617F мутације код појединачних протеина. Наведено повећање нивоа фактора инфламације указује да код оболелих од МПН постоји хронична инфламација. Третман хидроксиуреом и комбинацијом хидроксиурее и специфичног ЈАК2 инхибитора смањује нивое фактора инфламације у HEL 92.1.7 ћелијској линији, што указује да се испитивани S100 протеини експримирају на ЈАК2-завистан начин. Проинфламаторни цитокин IL-6 смањивао је нивое S100 протеина у мононуклеарима пацијената са МПН, а ово смањење код МПН посредовано је NF-κB и PI3K сигналним путевима. Антиинфламаторни цитокин IL-10 такође је смањивао нивое S100 протеина, поготово S100А8 и S100А9 код пацијената са ЕТ, али посредовано PI3K-АКТ сигналним путем. У IL-6 / IL-10 посредованој регулацији нивоа S100 протеина код МПН доминирају NF-κB и PI3K сигнални путеви, док је активација JAK-STAT сигналног пута конститутивна.Myeloproliferative neoplasms (MPNs) belong to the group of malignant diseases of the blood, characterized by a clonal expansion of neoplastically transformed hematopoietic progenitors of the myeloid lineage in the bone marrow. MPN in the narrow sense includes polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In patients with MPN, the underlying disorder is the JAK2V617F mutation, which is a driving mutation in more than 95% of patients with PV and 50-60% of patients with ET and PMF. The JAK2V617F mutation leads to constitutive activation of JAK2 kinase and cytokine-independent cell growth. As a consequence, downstream signaling pathways (JAK2 / STAT3, PI3K-AKT, MAPK), which are involved in myeloproliferation, are activated. The presence of the JAK2V617F mutation in MPN in hematopoietic cells alters the activity of inflammatory signaling pathways, altering the production of reactive oxygen species in neutrophils, altering serum inflammatory cytokine levels, and altering reactivity to these cytokines. The main feature of chronic inflammation is the high level of circulating cytokines and chemokines, as well as the accumulation of reactive oxygen species, which leads to genetic instability and may be suitable for the formation and progression of neoplasms. In patients with MPN, a significant increase in many inflammatory mediators has been observed, including a group of calcium-binding proteins, called S100 proteins. Due to their tumorigenic and pro-inflammatory activity, the S100 family of proteins that potentially have the greatest effect on the formation and development of myeloproliferative neoplasms are S100A4, S100A8, S100A9 and S100A12, as several studies have shown their elevated levels in MPN. The aim of this dissertation was to investigate the gene expression and levels of S100A4, S100A8, S100A9 and S100A12 factors in MPN patients, study the effect of pro-inflammatory cytokine IL-6, anti-inflammatory cytokine IL-10, as well as JAK inhibitors on the levels of these proteins on human HEL 92.1.7 model cell line with a JAK2 mutation, as well as an examination of the signaling mechanisms by which IL-6 and IL-10 exert their effects on peripheral blood cells of patients with MPN and the human HEL 92.1.7 model. cell line. The results of this study showed that inflammation factors show an increase in levels, which is dependent on the presence of the JAK2V617F mutation in individual proteins. This increase in the level of inflammatory factors indicates that patients with MPN have chronic inflammation. Treatment with hydroxyurea and a combination of hydroxyurea and a specific JAK2 inhibitor decreases the levels of inflammatory factor in the HEL 92.1.7 cell line, indicating that the S100 proteins examined are expressed in a JAK2-dependent manner. The pro-inflammatory cytokine IL-6 decreased S100 protein levels in mononuclear cells of patients with MPN, and this decrease in MPN was mediated by NF-κB and PI3K signaling pathways. The anti-inflammatory cytokine IL-10 also reduced S100 protein levels, especially S100A8 and S100A9 in patients with ET, but mediated by the PI3K-AKT signaling pathway. IL-6 / IL-10-mediated regulation of S100 protein levels by MPNs is dominated by NF-κB and PI3K signaling pathways, whereas activation of the JAK-STAT signaling pathway is constitutive

Variability of the chloroplast dna of sessile oak (Quercus petraea agg. Ehrendorfer, 1967) in Serbia

Genetic variability of sessile oak (Quercus petraea agg. Ehrendorfer, 1967) in Serbia is estimated applying cpDNA universal primer pairs that were characterized by a high informative level for chloroplast genome variability assessment in previous investigations. Five different haplotypes were detected in the analyzed sample material from populations in Serbia

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

Proliferation and differentiation markers of colorectal adenocarcinoma and their correlation with clinicopathological factors

Background/aim: The purpose of this study was to investigate proliferation and differentiation markers in colorectal adenocarcinoma and their correlation with clinicopathological factors. Materials and methods: Samples were collected from 38 patients with colorectal adenocarcinoma and 10 healthy controls. E-cadherin, carcinoembryonic antigen (mCEA), cyclin B1, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) receptor (EPOR) were examined by immunohistochemistry; VEGF and EPO were examined by real-time PCR. Results: The tumor samples were mostly characterized by large dimension (pT3), moderate level of differentiation (G2), negative lymph node status (N0), and no metastasis. Cyclin B1 and VEGF gene and protein expressions were significantly higher in tumor tissues than in control tissues; E-cadherin expression was significantly decreased in tumor samples and in positive correlation with mCEA. EPO was almost undetectable in tumor tissues of colorectal adenocarcinoma. Significant positive correlation was detected between tumor size and cyclin B1, tumor grade, and lymph node status. Conclusion: Decreased expression of EPO, high levels of VEGF and cyclin B1 expression, predominant moderate tumor differentiation, absence of metastasis, and negative lymph node status may suggest low level of aggressiveness, better prognosis, and longer patient survival

Regulation of S100As Expression by Inflammatory Cytokines in Chronic Lymphocytic Leukemia

The calcium-binding proteins S100A4, S100A8, and S100A9 are upregulated in chronic lymphocytic leukemia (CLL), while the S100A9 promotes NF-κB activity during disease progression. The S100-protein family has been involved in several malignancies as mediators of inflammation and proliferation. The hypothesis of our study is that S100A proteins are mediators in signaling pathways associated with inflammation-induced proliferation, such as NF-κB, PI3K/AKT, and JAK/STAT. The mononuclear cells (MNCs) of CLL were treated with proinflammatory IL-6, anti-inflammatory IL-10 cytokines, inhibitors of JAK1/2, NF-κB, and PI3K signaling pathways, to evaluate S100A4, S100A8, S100A9, and S100A12 expression as well as NF-κB activation by qRT-PCR, immunocytochemistry, and immunoblotting. The quantity of S100A4, S100A8, and S100A9 positive cells (p < 0.05) and their protein expression (p < 0.01) were significantly decreased in MNCs of CLL patients compared to healthy controls. The S100A levels were generally increased in CD19+ cells compared to MNCs of CLL. The S100A4 gene expression was significantly stimulated (p < 0.05) by the inhibition of the PI3K/AKT signaling pathway in MNCs. IL-6 stimulated S100A4 and S100A8 protein expression, prevented by the NF-κB and JAK1/2 inhibitors. In contrast, IL-10 reduced S100A8, S100A9, and S100A12 protein expressions in MNCs of CLL. Moreover, IL-10 inhibited activation of NF-κB signaling (4-fold, p < 0.05). In conclusion, inflammation stimulated the S100A protein expression mediated via the proliferation-related signaling and balanced by the cytokines in CLL

Hydroxyurea-induced senescent peripheral blood mesenchymal stromal cells inhibit bystander cell proliferation of JAK2V617F-positive human erythroleukemia cells

Hydroxyurea (HU) is a nonalkylating antineoplastic agent used in the treatment of hematological malignancies. HU is a DNA replication stress inducer, and as such, it may induce a premature senescence-like cell phenotype; however, its repercussion on bystander cell proliferation has not been revealed so far. Our results indicate that HU strongly inhibited peripheral blood mesenchymal stromal cells (PBMSC) proliferation by cell cycle arrest in S phase, and that, consequently, PBMSC acquire senescence-related phenotypical changes. HU-treated PBMSC display increased senescence-associated beta-galactosidase levels and p16(INK4) expression, as well as DNA damage response and genotoxic effects, evidenced by expression of gamma H2A.X and micronuclei. Moreover, HU-induced PBMSC senescence is mediated by increased reactive oxygen species (ROS) levels, as demonstrated by the inhibition of senescence markers in the presence of ROS scavenger N-acetylcysteine and NADPH oxidase inhibitor Apocynin. To determine the HU-induced bystander effect, we used the JAK2V617F-positive human erythroleukemia 92.1.7 (HEL) cells. Co-culture with HU-induced senescent PBMSC (HU-S-PBMSC) strongly inhibited bystander HEL cell proliferation, and this effect is mediated by both ROS and transforming growth factor (TGF)-beta expression. Besides induction of premature senescence, HU educates PBMSC toward an inhibitory phenotype of HEL cell proliferation. Finally, our study contributes to the understanding of the role of HU-induced PBMSC senescence as a potential adjuvant in hematological malignancy therapies

Inflammation Promotes Oxidative and Nitrosative Stress in Chronic Myelogenous Leukemia

Chronic inflammation is characterized by the production of reactive oxygen species (ROS), reactive nitrogen species, and inflammatory cytokines in myeloproliferative neoplasms (MPNs). In addition to these parameters, the aim of this study was to analyze the influence of ROS on the pro-liferation-related AKT/mTOR signaling pathway and the relationship with inflammatory factors in chronic myelogenous leukemia (CML). The activity of the antioxidant enzymes superoxide dis-mutase, glutathione peroxidase, and catalase is reduced in erythrocytes while levels of the oxidative stress markers malondialdehyde and protein carbonyl are elevated in the plasma of patients with CML. In addition, nitrogen species (nitrotyrosine, iNOS, eNOS) and inflammation markers (IL-6, NFkB, and S100 protein) were increased in granulocytes of CML while anti-inflammatory levels of IL-10 were decreased in plasma. CML granulocytes exhibited greater resistance to cytotoxic H2O2 activity compared to healthy subjects. Moreover, phosphorylation of the apoptotic p53 protein was reduced while the activity of the AKT/mTOR signaling pathway was increased, which was further enhanced by oxidative stress (H2O2) in granulocytes and erythroleukemic K562 cells. IL-6 caused oxidative stress and DNA damage that was mitigated using antioxidant or inhibition of inflammatory NFkB transcription factor in K562 cells. We demonstrated the presence of oxidative and ni-trosative stress in CML, with the former mediated by AKT/mTOR signaling and stimulated by in-flammation

VEGF Regulation of Angiogenic Factors via Inflammatory Signaling in Myeloproliferative Neoplasms

Background: Chronic inflammation has been recognized in neoplastic disorders, including myeloproliferative neoplasm (MPN), as an important regulator of angiogenesis. Aims: We investigated the influence of vascular endothelial growth factor (VEGF) and pro-inflammatory interleukin-6 (IL-6) on the expression of angiogenic factors, as well as inflammation-related signaling in mononuclear cells (MNC) of patients with MPN and JAK2V617F positive human erythroleukemic (HEL) cells. Results: We found that IL-6 did not change the expression of angiogenic factors in the MNC of patients with MPN and HEL cells. However, IL-6 and the JAK1/2 inhibitor Ruxolitinib significantly increased angiogenic factors—endothelial nitric oxide synthase (eNOS), VEGF, and hypoxia-inducible factor-1 alpha (HIF-1α)—in patients with polycythemia vera (PV). Furthermore, VEGF significantly increased the expression of HIF-1α and eNOS genes, the latter inversely regulated by PI3K and mTOR signaling in the MNC of primary myelofibrosis (PMF). VEGF and inhibitors of inflammatory JAK1/2, PI3K, and mTOR signaling reduced the eNOS protein expression in HEL cells. VEGF also decreased the expression of eNOS and HIF-1α proteins in the MNC of PMF. In contrast, VEGF increased eNOS and HIF-1α protein expression in the MNC of patients with PV, which was mediated by the inflammatory signaling. VEGF increased the level of IL-6 immunopositive MNC of MPN. In summary, VEGF conversely regulated gene and protein expression of angiogenic factors in the MNC of PMF, while VEGF increased angiogenic factor expression in PV mediated by the inflammation-related signaling. Conclusion: The angiogenic VEGF induction of IL-6 supports chronic inflammation that, through positive feedback, further promotes angiogenesis with concomitant JAK1/2 inhibition

Influence of inflammatory cytokines on S100A proteins expression in CLL patients

Background: S100A proteins possess a broad range of intra- and extracellular functions. The involvement of these proteins in inflammation-mediated responses is of particular interests, considering that inflammation represents one of the landmarks of malignancy. Processes such as inflammation and cellular stress trigger the release of S100A proteins to extracellular space, interacting with their receptors and activating numerous intracellular signaling pathways, for instance NF-κB and AP1. Through them, S100A proteins take part into regulation of some of the most essential cellular processes: cell differentiation, apoptosis, inflammation, proliferation, etc. Aims: The aim of the study is to assess the role of inflammation in activation of S100A proteins via proliferation and inflammation related signaling pathways. Methods: We observed 60 CLL patients’ samples to isolate mononuclear cells (MNC) and CD19+ cells. MNC of CLL patients were treated with pro-inflammatory IL-6 and anti-inflammatory IL-10 cytokines, and inhibitors of JAK1/2, NF-κB and PI3K signaling pathways, to evaluate S100A4, S100A8, S100A9, S100A12 and NF-κB protein expression by immunoblotting. Also, we used immunocytochemistry to analyse the number of the S100As immunopositive MNC of CLL patients.Results: S100A8 showed higher level of protein expression in MNC and CD19+cells in comparison to healthy control. The number of immunopositive S100A4 (p<0.05) and S100A9 cells (p<0.001) was signifi cantly decreased in CD19+cells and MNC, respectively of CLL patients in comparison to healthy control. In addition, S100A4 and S100A9 proteins expression had statistically significant lower level of expression in MNC. Also, IL-6 stimulated expression of S100A8 and S100A4 in MNC of CLL, while the expression of latter one was prevented by NF-κB and JAK1/2 inhibitors. IL-10 reduced expression of S100A8 and S100A12 in MNC of CLL.Summary/Conclusion: Pro-inflammatory IL-6 and anti-inflammatory IL-10 cytokines have opposite effect on inflammatory S100A8 protein in CLL, with a potential to be a prognostic marker