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The severity and mortality of coronavirus disease 2019 (COVID-19) are greater in males than in females, though the infection rate is the same in the two sexes. We investigated sex hormone differences associated with the hyperinflammatory immune response to SARS-CoV-2 on the basis of patients’ cytokine profiles and vaccination statuses. Clinical and laboratory data of 117 patients with COVID-19 were collected to examine sex differences associated with oxidative stress markers, neutrophil extracellular traps (NETs), and plasma cytokine levels up to 5 months from hospital admission. The testosterone and free testosterone levels were low in male patients with COVID-19 and returned to normal values after recovery from the disease. The dihydrotestosterone (DHT) levels were transiently reduced, while the sex hormone-binding globulin levels were decreased in post-COVID-19 male patients. The levels of the inflammatory cytokines interleukin-6 (IL-6) and IL-10 appeared generally increased at diagnosis and decreased in post-COVID-19 patients. In females, the concentration of tumor necrosis factor-alpha was increased by four times at diagnosis. The levels of the coagulation markers intercellular adhesion molecule-1 (ICAM-1) and E-selectin were consistently upregulated in post-COVID-19 female patients, in contrast to those of vascular cell adhesion molecule-1 (VCAM-1), P-selectin, and chemokine IL-8. DHT increased the levels of reactive oxygen species in the neutrophils of male patients, while estradiol decreased them in females. Markers for NET, such as circulating DNA and myeloperoxidase, were significantly more abundant in the patients’ plasma. Sex hormones have a potential protective role during SARS-CoV-2 infection, which is weakened by impaired testosterone synthesis in men.

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.

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.

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.

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.

Background: Oxidative stress is caused by imbalance between excessive production of reactive oxygen species and decreased capabilities of antioxidant system, and it is recognized as a feature in cancerogenesis, as well in hematologic malignancies. Previous studies have shown increasing expression of oxidative stress markers and antioxidant enzymes in lymph nodes progressing to aggressive lymphomas. Aims: The aim of our study was to assess the clinical and prognostic significance of oxidative stress markers in patients with untreated diffuse large B-cell lymphoma (DLBCL). Methods: We analysed 64 patients diagnosed with DLBLC during 2018 and 2019, while 27 healthy volunteers (51.9% males) served as a control group. The plasma sample and laboratory analyses were obtained prior to initiation of specific hematologic treatment. After completion of the therapy, the patients were followed up for up to 4 years and for each of them progression free survival (PFS) and overall survival (OS) were calculated. Malondialdehyde (MDA) and protein carbonyl (PC) were used as markers of oxidative stress in plasma of patients and volunteers, while catalase is used as an antioxidant marker. Results: The mean patients’ age was 56.2 years (range, 20–87); 51.6% were males. Majority of patients were analysed before 1L therapy (n=61; 95,3%), and had following clinical stages: Ann Arbor stage I 23.4%, stage II 37.5%, stage III 15.6% and stage IV 23.4%. Majority of the patients had satisfactory performance status (73.5% had ECOG PS ≥1), bulky tumorous mass was present in 34.4% of patients, whereas 70.3% had extranodal localisation of lymphoma. MDA (6.66±2.7 nmol/ml) was significantly increased (p<0.001, 2.6-fold), while PC (4.29±2.7 nmol/mg) was also significantly increased (p=0.0027, 5-fold) in patients with DLBCL compared to healthy volunteers. In opposite, antioxidant catalase (0.194±0.06 IU/ml) was significantly reduced (p=0.0034, 1.9-fold) in patients with DLBCL. MDA was in significant (p<0

Background:: Hydroxyurea (HU) is a chemotherapeutic agent that reduces ribonucleotide reductase, stops DNA synthesis and repar, and therefore causes cell proliferation inhibition and apoptosis. Due to its cytostatic properties, HU is frequently used for treatment of myeloproliferative neoplasms, ovarian cancer, and sickle cell anemia. Nitric oxide (NO), produced by nitric oxide synthase (NOS) enzymes is a potent signaling molecule involved in blood flow regulation, neutrotransmission, and immunity. Although HU treatment increases NO levels, up to date it is not clear whether it originates from activation of NOS enzymes or HU degradation. Aims: The aim of this study was to determine the involvement of NOS2 enzyme in the cytostatic effect of HU. Methods: To examine the involvement of the NOS2 enzyme in the molecular mechanism of HU, we treated erythroleukemic HEL92.7.1 cells with pan-selective NOS inhibitor L-NAME (200µM, 1mM, and 5mM), NOS2 specific inhibitor 1400W (1, 10, and 100µM), or NOS2/NOS3 inhibitor DPI (1, 5, and 10µM), in combination with hydroxyurea (200µM), and monitored their effect on proliferation and cell cycle. Immunocytochemistry for the proliferation marker Ki67 was performed to assess proliferation, while cell distribution in cell cycle phases was determined by flow cytometry after propidium iodide staining. Colony forming assay have been performed with the bone marrow cells of Nos2 null mice after oral HU treatment to corroborate the data obtained by enzymatic inhibition. Results: In this study, we demonstrated that treatment of HEL92.7.1 cells with HU induces a dose-dependent increase in NOS2 protein levels and two products of the enzyme NOS - NO and citrulline. HU-induced citrulline levels can be reduced by treatment with the NOS inhibitor L-NAME, indicating that NO is produced de novo by the NOS enzyme rather than HU degradation. Inhibition of the NOS2 enzyme by L-NAME, 1400W, or DPI was sufficient to abolish HU-mediated inhibition of prolifer

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, 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 ekspr

Myeloproliferative neoplasms (MPNs) are developing resistance to therapy by JAK1/2 inhibitor ruxolitinib. To explore the mechanism of ruxolitinib's limited effect, we examined the JAK1/2 mediated induction of proliferation related ERK1/2 and AKT signaling by proinflammatory interleukin-6 (IL-6) in MPN granulocytes and JAK2V617F mutated human erythroleukemia (HEL) cells. We found that JAK1/2 or JAK2 inhibition prevented the IL-6 activation of STAT3 and AKT pathways in polycythemia vera and HEL cells. Further, we showed that these inhibitors also blocked the IL-6 activation of the AKT pathway in primary myelofibrosis (PMF). Only JAK1/2 inhibitor ruxolitinib largely activated ERK1/2 signaling in essential thrombocythemia and PMF (up to 4.6 fold), with a more prominent activation in JAK2V617F positive granulocytes. Regarding a cell cycle, we found that IL-6 reduction of HEL cells percentage in G2M phase was reversed by ruxolitinib (2.6 fold). Moreover, ruxolitinib potentiated apoptosis of PMF granulocytes (1.6 fold). Regarding DNA replication, we found that ruxolitinib prevented the IL-6 augmentation of MPN granulocytes frequency in the S phase of the cell cycle (up to 2.9 fold). The inflammatory stimulation induces a cross-talk between the proliferation linked pathways, where JAK1/2 inhibition is compensated by the activation of the ERK1/2 pathway during IL-6 stimulation of DNA replication.

In accordance with increased proliferation in myeloproliferative neoplasm (MPN), the goal is to evaluate the immunoexpression of: beta-catenin, PPAR-gamma and Ki67 protein, to compare them with bone marrow ultrastructural characteristics in patients with MPN. Immunoexpression and electron microscopy of bone marrow was analyzed in 30 Ph-negative MPN patients, including per 10 patients with polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The quantity of beta-catenin immunoreactive cells was significantly higher in PV then in ET (p lt 0.01) or PMF group of patients (p lt 0.01) and also in ET versus PMF group of patients (p lt 0.01). Erythroid lineage showed absent beta-catenin staining without immunoreactivity in nucleus. In contrast, immunoreactivity for PPAR-gamma was localized mostly in megakaryocytes and the highest number of PPAR-gamma immunopositive cells was detected in PMF group of patients. In addition, the proliferative Ki67 index was significantly increased in the PMF and PV patients compared to patients with ET. Also, the megakaryocytes showed abnormal maturation in PMF group of patients as determined by ultrastructural analysis. These results indicated that PV dominantly expressed beta-catenin and proliferation marker Ki67 in bone marrow, while PMF is linked preferentially to PPAR-gamma immunopositive megakaryocytes characterized by abnormal maturation.

This study has been performed to determine the mechanism of activation of the myeloid related S100A proteins by inflammatory cytokines in myeloproliferative neoplasm (MPN). Besides microarray analysis of MPN-derived CD34(+) cells, we analysed the pro-inflammatory IL6 and anti-inflammatory IL10 dependence of NF-kappa B, PI3K-AKT, and JAK-STAT signalling during induction of S100A proteins in mononuclear cells of MPN, by immunoblotting and flow cytometry. We observed the reduced gene expression linked to NF-kappa B and inflammation signalling in MPN-derived CD34(+) cells. Both IL6 and IL10 reduced S100A8 and 100A9 protein levels mediated via NF-kappa B and PI3K signalling, respectively, in mononuclear cells of essential thrombocythemia (ET). We also determined the increased percentage of S100A8 and S100A9 positive granulocytes in ET and primary myelofibrosis, upgraded by the JAK2V617F mutant allele burden. S100A8/9 heterodimer induced JAK1/2-dependent mitotic arrest of the ET-derived granulocytes. Significance of the study We demonstrated that inflammation reduced the myeloid related S100A8/9 proteins by negative feedback mechanism in ET. S100A8/9 can be a diagnostic marker of inflammation in MPN, supported by the concomitant NF-kappa B and JAK1/2 signalling inhibition in regulation of myeloproliferation and therapy of MPN.

Epithelial to mesenchymal transition (EMT) is a feature of several types of human cancer, including oral squamous cell carcinoma (OSCC). In the present study, tumor and margin cell cultures obtained from patients with OSCC were used to determine the expression patterns of certain EMT-associated markers, including vimentin, alpha -smooth muscle actin, SLUG and SNAIL. In addition, other EMT-associated features, including clonal, proliferative and migratory potential were compared between the two cell types. Cell cultures were generated from tumor and margin tissue samples from 6 patients and cultured up to the fifth passage. EMT marker expression was assessed by reverse transcription-quantitative PCR. Cell proliferation, colony formation and scratch wound healing assays were conducted to characterize the two cell types in terms of proliferation rates, clonality and motility. All of the studied markers were expressed in tumor and margin cells. Although no significant differences were noted with regard to the aforementioned markers, their expression tended to be higher in margin cultures than in tumor cultures. The expressions of the EMT markers were also higher in the fifth passage compared with those noted at the first with a few exceptions. The rates of proliferation and cell migration were decreased during passages, while the number of colonies was increased in both types of cell culture. Tumor and margin cells indicated certain similarities with regard to EMT transition characteristics.

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.

Purpose: A common feature of malignancies is increased reactive oxygen species (ROS) and reactive nitrogen species (RNS). We analyzed the influence of oxidative and nitrosative stress on the activation of AKT/mTOR signaling pathway in myeloproliferative neoplasms (MPN). Methods: Oxidative stress-induced gene expression in circulatory CD34(+) cells of MPN patients was studied by microarray analysis. Biomarkers of oxidative and nitrosative stress were determined using spectrophotometry in plasma and erythrocyte lysate. The levels of nitrotyrosine, inducible NO synthase (iNOS) and AKT/mTOR/p70S6K phosphorylation were determined by immunocytochemistry and immunoblotting in granulocytes of MPN patients. Results: Antioxidants superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) gene expression were increased in circulatory CD34(+) cells, while SODI and GPx enzymes were reduced in the erythrocytes of MPN. Plasma malonyl-dialdehyde and protein carbonyl levels were elevated in MPN. The total antioxidant capacity in plasma and erythrocyte catalase (CT) activities was the most prominent in primary myelofibrosis (PMF) with JAK2V617F heterozygosity. The total nitrite/ nitrate (NOx) level was augmented in the plasma of PMF patients (p lt 0.001), while nitrotyrosine and iNOS were generally increased in the granulocytes of MPN patients. Activation of AKT/m TOR signaling was the most significant in PMF (p lt 0.01), but demonstrated JAK2V617F dependence and consequent p70S6K phosphorylation in the granulocytes of essential thrombocytemia (ET) and polycythemia vera (PV) patients. Hydrogen peroxide stimulated mTOR pathway, iNOS and nitrotyrosine quantities, the last one prevented by the antioxidant nacetyl-cysteine (NAC) in the granulocytes of MPN. Conclusion: Our study showed increased levels of oxidative and nitrosative stress parameters in MPN with JAK2V617F dependence. The ROS enhanced the constitutive activation of AKT/mTOR signaling and nitrosative parameters in MPN.

Increased angiogenesis in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) has been recognized, but its connection with clinical and molecular markers needs to be defined. The aims of study were to (1) assess bone marrow (BM) angiogenesis measured by microvessel density (MVD) using CD34 and CD105 antibodies; (2) analyze correlation of MVD with plasma angiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8; (3) examine the association of MVD with clinicopathological and molecular markers. We examined 90 de novo MPN patients (30 polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET)) and 10 age-matched controls. MVD was analyzed by immunohistochemistry "hot spot" method, angiogenic factors by immunoassay and JAK2V617F, and CALR mutations by DNA sequencing and allelic PCR. MVD was significantly increased in MPNs compared to controls (PMF gt PV gt ET). Correlation between MVD and plasma angiogenic factors was found in MPNs. MVD was significantly increased in patients with JAK2V617F mutation and correlated with JAK2 mutant allele burden (CD34-MVD: rho = 0.491, p lt 0.001; CD105-MVD: rho = 0.276, p = 0.02) but not with CALR mutation. MVD correlated with leukocyte count, serum lactate dehydrogenase, hepatomegaly, and splenomegaly. BM fibrosis was significantly associated with CD34-MVD, CD105-MVD, interleukin-8, and JAK2 mutant allele burden. JAK2 homozygote status had positive predictive value (100%) for BM fibrosis. Patients with prefibrotic PMF had significantly higher MVD than patients with ET, and we could recommend MVD to be additional histopathological marker to distinguish these two entities. This study also highlights the strong correlation of MVD with plasma angiogenic factors, JAK2 mutant allele burden, and BM fibrosis in MPNs.