Analiza ekspresije eNOS i angiogenih faktora HIF-1alfa i VEGF u mijeloproliferativnim neoplazmama: veza sa prisustvom mutacija u genima za JAK2 i CALR

hematopoiesis characterized by a disorder of proliferation of one or more myeloid cell lines. MPNs include 3 entities: polycythemia (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). The most common disorder in MPN is the JAK2V617F mutation present in 97% of patients diagnosed with PV and 50-60% of patients with ET and PMF diagnoses. The JAK2V617F mutation induces the constituent activation of the innate signal pathways (JAK2 / STAT3, PI3K / AKT, MAPK) involved in myeloproliferation. The second most common somatic mutation, which is present in most JAK2V617F negative ET and PMF patients, is associated with the calreteculin (CALR) encoding genome present in 31.9% ET and 31.7% of PMF patients. In the case of various hematological malignancies, including MPN, as a reflection of increased angiogenesis, the presence of elevated expression of vascular endothelial growth factor (VEGF) has been described. Recent studies have shown the relationship between the expression of the VEGF gene and the levels of hypoxia-inducible factor-1 (HIF-1) and nitrogen monoxide (NO). Several studies have so far shown that NO can induce the production of VEGF via PI3K / Akt / PKB / HIF-1α times, but also acts as an inhibitor of HIF-1α expression. The aim of the study was to investigate the degree of expression and correlation of key angiogenic factors - HIF-1α, VEGF and endothelial NO synthase (eNOS) in the bone marrow, CD34+ cells and granulocytes of MPN patients according to JAK2 and CALR status and therapy. In addition, we used proinflammatory IL-6 to induce the expression of HIF-1α, VEGF and eNOS in the human HEL 92.1.7 cell line with a JAK2 mutation, as well as in differentiated macrophages. Also studied was the participation of various signaling pathways (JAK2 / STAT3, PI3K / Akt, MAPK) through which IL-6 achieved its effects on the HEL cell line model as well as in granulocyte of MPN patients. The results of this study have shown that angiogenic factors have a more pronounced expression in granulocytes than in CD34+ and bone marrow cells, which indicates the importance of inflammatory granulocytes in the development of angiogenesis. A significant negative correlation between HIF-1α and VEGF protein expression, as well as HIF-1α and eNOS, was shown, while there was a significant positive correlation between VEGF and eNOS in granulocytes of MPN patients. Standard treatment with hydroxyurea in MPN patients has antiangiogenic potential in terms of reducing all three angiogenic factors - HIF-1α, VEGF and eNOS in granulocytes...Mijeloproliferativne neoplazme (MPN) predstavljaju klonalne bolesti matičnih ćelija hematopoeze koje karakteriše poremećaj proliferacije jedne ili više mijeloidnih ćelijskih linija. MPN obuhvataju 3 entiteta: policitemiju veru (PV), esencijalnu trombocitemiju (ET) i primarnu mijelofibrozu (PMF). Najzastupljeniji poremećaj kod MPN je JAK2V617F mutacija koja je prisutna kod 97% pacijenata sa dijagnozom PV i 50-60% pacijenata sa dijagnozama ET i PMF. JAK2V617F mutacija indukuje konstitutivnu aktivaciju nishodnih signalnih puteva (JAK2/STAT3, PI3K/AKT, MAPK) koji su uključeni u mijeloproliferaciju. Druga najzastupljenija somatska mutacija, koja je prisutna kod većine JAK2V617F negativnih ET i PMF pacijenata, povezana je sa genom koji kodira kalretikulin (CALR) prisutna kod 31,9% ET i 31,7% PMF pacijenata. Kod različitih hematoloških maligniteta, uključujući i MPN, kao odraz pojačane angiogeneze, opisano je prisustvo povišene ekspresije faktora rasta vaskularnog endotela (VEGF). U novijim istraživanjima pokazana je veza između ekspresije VEGF gena i nivoa hipoksija-inducibilnog faktora-1 (HIF-1) azot monoksida (NO). Nekoliko studija do sada je pokazalo da NO može indukovati produkciju VEGF putem PI3K/Akt/PKB/HIF-1α puta, ali i da deluje kao inhibitor HIF-1α ekspresije. Cilj istraživanja ove disertacije bilo je ispitivanje stepena ekspresije i korelacija ključnih angiogenih faktora - HIF-1α, VEGF i endotelne NO sintaze (eNOS) u kostnoj srži, CD34+ ćelijama i granulocitima MPN pacijenata prema JAK2 i CALR statusu i terapiji. Pored toga, koristili smo proinflamatorni IL-6 da indukujemo ekspresiju HIF-1α, VEGF i eNOS u humanoj HEL 92.1.7 ćelijskoj liniji sa JAK2 mutacijom, kao i u diferentovanim makrofagama. Takođe ispitivano je učešće različitih signalnih puteva (JAK2/STAT3, PI3K/Akt, MAPK) putem kojih IL-6 ostvaruje svoje efekte, na modelu HEL ćelijske linije kao i kod granulocita MPN pacijenata. Rezultati ove studije su pokazali da angiogeni faktori imaju izraženiju ekspresiju u granulocitima nego u CD34+ i ćelijama kostne srži, što ukazuje na značaj inflamatornih granulocita u razvoju angiogeneze. Pokazana je i značajna negativna korelacija između proteinske ekspresije HIF-1α i VEGF, kao i HIF-1α i eNOS, dok između VEGF i eNOS postoji značajna pozitivna korelacija kod granulocita MPN pacijenata. Standardno lečenje primenom hidroksiureje kod MPN pacijenata ima antiangiogeni potencijal u smislu smanjenja sva tri angiogena faktora – HIF-1α, VEGF i eNOS u granulocitima..

Nitric Oxide Synthase Dependency in Hydroxyurea Inhibition of Erythroid Progenitor Growth

Hydroxyurea (HU) causes nitric oxide (NO) bioactivation, acting as both a NO donor and a stimulator of NO synthase (NOS). To examine whether HU effects are NO mediated by chemical degradation or enzymatic induction, we studied human and mouse erythroid cells during proliferation, apoptosis, and differentiation. The HU and NO donor demonstrated persisted versus temporary inhibition of erythroid cell growth during differentiation, as observed by γ-and β-globin gene expression. HU decreased the percentage of erythroleukemic K562 cells in the G2/M phase that was reversed by N-nitro l-arginine methyl ester hydrochloride (L-NAME). Besides activation of endothelial NOS, HU significantly increased apoptosis of K562 cells, again demonstrating NOS dependence. Administration of HU to mice significantly inhibited colony-forming unit-erythroid (CFU-E), mediated by NOS. Moreover, burst-forming-units-erythroid (BFU-E) and CFU-E ex vivo growth was inhibited by the administration of nitrate or nitrite to mice. Chronic in vivo NOS inhibition with L-NAME protected the bone marrow cellularity despite HU treatment of mice. NO metabolites and HU reduced the frequency of NOS-positive cells from CFU-E and BFU-E colonies that was reverted by NOS inhibition. HU regulation of the G2/M phase, apoptosis, differentiation, cellularity, and NOS immunoreactive cells was NOS dependent. Inhalation of NO therapy as well as strategies to increase endogenous NO production could replace or enhance HU activity

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

Hydroxyurea Induces Bone Marrow Mesenchymal Stromal Cells Senescence and Modifies Cell Functionality In Vitro

Hydroxyurea (HU) is an antineoplastic agent that functions as an antimetabolite compound by inhibiting the ribonucleotide reductase. HU acts mainly as a cytostatic drug that through DNA replication stress may trigger a premature senescence-like cell phenotype, though its influence on bone marrow-derived mesenchymal stem/stromal cell (BMMSC) functions has not elucidated yet. Our results indicate that HU inhibits the growth of human BMMSC alongside senescence-like changes in both morphology and replicative potential, provokes cell cycle arrest at the S phase without affecting cellular viability and induces the expression of senescence-associated β-galactosidase and p16INK4. Moreover, HU-induced senescent BMMSC, although they did not change MSC markers expression, exhibited reduced capacity osteogenic and adipogenic differentiation. Conversely, HU treatment increased immunoregulatory functions of BMMSC compared with untreated cells and determined by T-cell proliferation. Interestingly, HU did not influence the capacity of BMMSC to induce monocytic myeloid-derived suppressor cells. Thus, these results suggest that HU improves the BMMSC functions on the T-cell inhibition and preserves their interaction with myeloid cell compartment. Mechanistically, BMMSC under HU treatment displayed a downregulation of mTOR and p38 MAPK signaling that may explain the reduced cell differentiation and increased immunomodulation activities. Together, the results obtained in this investigation suggest that HU by inducing senescence-like phenotype of BMMSC influences their cellular differentiation and immunoregulatory functions

Nitric Oxide Mediation in Hydroxyurea and Nitric Oxide Metabolites’ Inhibition of Erythroid Progenitor Growth

In several systems, hydroxyurea has been shown to trigger nitric oxide (NO) release or activation of NO synthase (NOS). To elucidate this duality in its pharmacological effects, during myelosuppression, we individually examined hydroxyurea’s (NO releasing agent) and NO metabolites’ (stable NO degradation products) effects on erythroid colony growth and NOS/NO levels in mice using NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Hydroxyurea and nitrite/nitrate decreased the bone marrow cellularity that was blocked by PTIO only for the NO metabolites. Hydroxyurea inhibition of colony-forming unit-erythroid (CFU-E) formation and reticulocytes was reversed by PTIO. Moreover, hydroxyurea, through a negative feedback mechanism, reduced inducible NOS (iNOS) expressing cells in CFU-E, also prevented by PTIO. Nitrate inhibition of burst-forming units-erythroid (BFU-E) colony growth was blocked by PTIO, but not in mature CFU-E. The presented results reveal that NO release and/or production mediates the hydroxyurea inhibition of mature erythroid colony growth and the frequency of iNOS immunoreactive CFU-E

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

Gender Difference in SARS-CoV-2 Stimulation of Hyperinflammatory Response in Patients with COVID-19

Background: Although women and men have the same prevalence, men with COVID-19 are more at risk for worse outcomes and death, independent of age. Methods: To observe the hyperinflammatory response regarding sex, we will measure the levels of inflammatory cytokines: interleukin-6 (IL-6), IL-1β, tumor necrosis factor-alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), IL-10, interferon- gamma (IFN-γ), IL-8, transforming growth factor-beta 1 (TGF-β1), analyzed by ELISA, in plasma of 130 COVID-19 patients at diagnosis and correlate them with clinical parameters. Besides, we checked the quality of life of patients up to 3 months after hospitalization. Results: Pro-inflammatory IL-6 was significantly (p<0.01) increased in male COVID-19 patients compared to healthy male volunteers (4.7-fold) and female COVID-19 patients (1.75-fold). IL-6 was positively correlated to INR, aPTT (p<0.001), and ferritin (p<0.05), while INR with CRP (p<0.05). IL-8 was significantly increased in female COVID-19 patients compared to healthy female volunteers (p<0.01, 2.5-fold) and male COVID-19 patients (p<0.05). TNF-α was significantly (p<0.05) increased in male COVID-19 patients compared to healthy male volunteers. MCP-1 and IFN-γ were significantly increased in female COVID-19 patients compared to healthy female volunteers (p<0.01). Anti-inflammatory TGF-β1 was decreased in COVID-19 patients regardless of gender. Most patients were men (75%) with chronic disorders (59%, mostly hypertension). COVID-19 reduced their social (32%) and physical activities (34%) after 6 weeks of diagnosis, but further on reduced in additional 3 months (social 55%) with depression (22%). Conclusions: Inflammatory response is generally increased in COVID-19 patients with specific cytokines in accordance with gender

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