Clear-cell renal cell carcinoma (ccRCC) is associated with the mutated von Hippel–Lindau (VHL) gene leading to the activation of hypoxia-inducible factor 1A (HIF1A) and subsequent overexpression of erythropoietin (EPO). We analyzed tumor and healthy tissues from 43 ccRCC patients after radical nephrectomy and cultured 786-O (biallelic VHL inactivation) and Caki-1 (wild-type VHL) cells in normal (21% O2) and low oxygen (3% O2) with 10% and 2% fetal bovine serum (FBS). DNA sequencing, including Sanger sequencing, MLPA and LOH, revealed 27 somatic mutations of VHL in ccRCC. HIF1A protein showed decreased or no expression in tumors compared to healthy tissue, independent of VHL alteration. The 786-O cells showed increased HIF1A protein expression after 48 h under low oxygen and 10% FBS. EPO and erythropoietin receptor (EPOR) were significantly decreased in ccRCC without HIF1A expression. EPO mRNA increased in the 786-O cells at 3% O2 after 48 h, while the Caki-1 cells had low or no EPO expression. Hypoxia increased EPOR mRNA in the Caki-1 cells at 10% FBS, but decreased in the 786-O cells at 2% FBS after 48 h. JAK2/STAT5A activity was increased only in HIF1A-positive tumors. These results suggest that EPO/EPOR activation in ccRCC is mainly driven by low oxygen, not VHL regulation of hypoxia-related responses.

Abstract Background Male sex is associated with greater severity and mortality from coronavirus disease 2019 (COVID-19), although infections are equally distributed between men and women. The present study aims to investigate sex differences in the hyperinflammatory immune response to SARS-CoV-2 infection and consequent thrombosis using linked cytokine profile and blood laboratory data. Methods This observational cohort study involved 99 COVID-19 patients (69 male and 30 female adults), hospitalized between March 2021 and April 2022. Their clinical and laboratory data were collected to examine sex difference in oxidative stress, neutrophil extracellular traps (NETs) formation and plasma cytokines at hospital admission and up to 5 months of their recovery. Results Dihydrotestosterone (DHT) levels were transiently reduced, while sex hormone binding globulin levels were continuously decreased in male post-COVID-19 patients after its rise at diagnosis. Regarding inflammatory cytokines, interleukin-6 (IL-6) and interferon-gamma are generally increased at diagnosis, while IL-6 is decreasing in post-COVID-19 patients. Tumor necrosis factor-alpha had 5-fold increase in females at diagnosis. Chemokines IL-8 and monocyte chemoattractant protein-1 and coagulation markers intercellular adhesion molecule-1 and E-selectin were consistently upregulated in female COVID-19 and post-COVID-19 patients, in contrast to vascular cell adhesion molecule-1 and P-selectin. DHT increased reactive oxygen species in neutrophils of male patients, while estrogen decreased in females. Markers for NETs formation, such as circulating DNA and myeloperoxidase, were significantly increased in the plasma of patients. Sex hormones are mostly negatively correlated with leukocytes, while in positive correlation with coagulation markers. Conclusions Markers of chemotaxis, endothelial dysfunction and inflammation are still detectable and partially sex dependent in COVID-19 patients after 5 months of hospital admission. These distributions of sex steroid hormones splitted endothelial adhesion molecules, coagulation and inflammation factors, while they generalized chemokines.

Context: Adrenal lesions are frequent among patients with sporadic neuroendocrine tumors (spNETs) or multiple endocrine neoplasia type 1 (MEN1). Armadillo repeat-containing 5 (ARMC5)-inactivating variants cause adrenal tumors and possibly other neoplasms. Objective: The objective of this work is to investigate a large cohort spNETs or MEN1 patients for changes in the ARMC5 gene. Patients and Methods: A total of 111 patients, 94 with spNET and 17 with MEN1, were screened for ARMC5 germline alterations.Thirty-six tumors (18 spNETs and 18 MEN1 related) were collected from 20 patients. Blood and tumor DNA samples were genotyped using Sanger sequencing and microsatellite markers for chromosomes. ARMC5 and MEN1 expression were assessed by immunohistochemistry. Results: In 76 of 111 (68.4%) patients, we identified 16 different ARMC5 germline variants, 2 predicted as damaging. There were no differences in the prevalence of ARMC5 variants depending on the presence of MEN1-related adrenal lesions. Loss of heterozygosity (LOH) at chromosome 16p and ARMC5 germline variants were present together in 23 or 34 (67.6%) tumors; in 7 of 23 (30.4%) their presence led to biallelic inactivation of the ARMC5 gene.The latter was more prevalent in MEN 1-related tumors than in spNETs (88.9% vs 38.9%; P = .005). LOH at the chromosome 16p (ARMC5) and 11q (MEN1) loci coexisted in 16/18 MEN1-related tumors, which also expressed lower ARMC5 (P= .02) and MEN1 (P= .01) proteins compared to peritumorous tissues. Conclusion: Germline ARMC5 variants are common among spNET and MEN1 patients. ARMC5 haploinsufficiency or biallelic inactivation in spNETs and MEN1-related tumors suggests that ARMC5 may have a role in modifying the phenotype of patients with spNETs and/or MEN1 beyond its known role in macronodular adrenocortical hyperplasia.

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.

Introduction: Philadelphia-negative myeloproliferative neoplasms (Ph-MPN) are characterized by overproduction of one or more blood cell lines.Methods: We studied the proliferative characteristics of 91 patients with de novo Ph-MPN. Colony-forming cells (CFC) and endogenous colonies (EC), from bone marrow (BM) and/or peripheral blood (PB), were analyzed by colony assay based on methylcellulose. The level of circulating CD34(+) cells was determined by flow cytometry.Results: The total number of PB CFC in primary myelofibrosis (PMF) was increased compared to the control sample (P < 0.01) and essential thrombocythemia (ET) (P < 0.05). The highest number of BM and PB EC was observed in polycythemia vera (PV) (P < 0.01). Increased levels of CD34(+) cells characterized early-prefibrotic (57%) and advanced-fibrotic PMF (90%) as compared to PV (34%) and ET (32%) (P < 0.01). In the whole Ph-MPN group, the total number of PB CFC (P < 0.01), PB EC (P < 0.05), and CD34+ cells (P < 0.01) correlated with the degree of BM fibrosis. Higher levels of circulating CD34(+) cells in PMF correlated with the total number of PB EC (P < 0.05) and degree of BM fibrosis (P < 0.01).Conclusions: Exploration of the PB proliferative characteristics of Ph-MPN on diagnosis may be helpful in revealing early-prefibrotic PMF. Monitoring the levels of circulating CD34(+) cells may provide a sensitive indicator of fibrotic evolution in PV and PMF.

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF‐1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF‐1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34+ cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFβ and MAPK signaling pathways were detected in CD34+ cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation‐related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors. © 2016 Wiley Periodicals, Inc.

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.

Alterations in von Hippel-Lindau gene (VHL) do not determine deregulation of hypoxia-inducible factors (HIFs) in clear-cell renal carcinoma (ccRCC). Their effects on tuberous sclerosis proteins (TSC1/2) and heat shock protein 90 (Hsp90) expressions in sporadic ccRCC are unknown. Therefore, we analyze the impact of VHL alterations and HIF-α production on the expression of TSC proteins and Hsp90 in these tumors. Alterations in VHL gene region exhibited 37/47 (78.7%) tumors. Monoallelic inactivation (intragenic mutation or LOH) was found in 10 (21.3%) and biallelic inactivation (intragenic mutation plus LOH) in 27 (57.4%) ccRCCs. Tumorous expression of HIF-α mRNAs, HIF-α, Hsp90 and TSC2 were VHL independent; TSC2 was underexpressed in all tumors by immunostaining (P < 0.001). Immunoblotting revealed that TSC1 production was lower in tumors with monoallelic VHL inactivation than in control (P = 0.01) and tissues with biallelic VHL inactivation (P = 0.019), while tumors lacking HIF-1α (16/47) concurrently overexpressed HIF-2α and underexpressed TSC1 in comparison to controls (P = 0.01 for both) and HIF-1α positive tumors (P = 0.015 and P = 0.050). Significant portion of variability (56.4%) in tumor diameter was explained by oscillations in nuclear grade, and TSC1 and HIF-2α expression in VHL altered tumors. In conclusion, while TSC2 is broadly downregulated in sporadic ccRCC, TSC1 expression is reduced in two subsets of these tumors, those with monoallelic VHL gene inactivation and those with concurrent low HIF-1α and high HIF-2α expression. Hence, the involvement of nuclear grade, TSC1 and HIF-2α in the progression of VHL altered tumors, implies the interplay between pVHL and TSC1.

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34+ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34+ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34+ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34+ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34+ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34+ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.

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 JAK2 V617F mutation positive versus negative primary myelofibrosis (PMF) patients. Moreover, thrombocytosis was reduced by JAK2 V617F 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 JAK2 V617F allele burden; CSF3R, IL6ST , and STAT1/2 in ET and PV with JAK2 V617F 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 JAK2 V617F mutation presence in ET and PMF patients. Therefore, the JAK2 V617F mutant allele burden participated in inflammation biomarkers induction and related signaling pathways activation in MPNs.

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.

The blood flow from the placenta to the fetus depends on human umbilical vein (HUV) vascular tone. ATP-sensitive K+ (K-ATP) channels link the metabolic state of the cell to membrane potential, and their activation in the HUV represents protection against hypoxia. The aims of our study were to assess the effects of resveratrol and naringenin on the HUV and to define the roles of K-ATP channels in their effects. Serotonin or 100 mM K+ were used for precontraction of the HUV without endothelium. The cumulative concentration-response curves were obtained by adding increasing concentrations of resveratrol or naringenin. Glibenclamide was used, in order to test the role of K-ATP channels in its effect. Resveratrol induced more potent vasodilatation of serotonin- and 100 mM K+-precontracted HUV than naringenin. Glibenclamide induced significant shift to the right of the concentration-response curves of resveratrol and P1075 (a specific opener of K-ATP channels). Western blotting showed that HUV expressed protein Kir6.1. Thus, resveratrol and naringenin produce dilatation of HUV. It seems that K-ATP channels are involved in the relaxation of HUV induced by resveratrol, while naringenin seems to interact with other ion channels. The K+ channel-independent mechanism(s) of these polyphenols could not be excluded. Copyright (C) 2014 John Wiley & Sons, Ltd.

Purpose: The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation.Methods: We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71(+) cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation.Results: The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells.Conclusion: This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources.

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 μM 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 μM 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 Akt 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.