Vladan P. Cokic

Hydroxyurea induces fetal hemoglobin by the nitric oxide–dependent activation of soluble guanylyl cyclase

Hydroxyurea treatment of patients with sickle-cell disease increases fetal hemoglobin (HbF), which reduces hemoglobin S polymerization and clinical complications. Despite its use in the treatment of myeloproliferative diseases for over 30 years, its mechanism of action remains uncertain. Recent studies have demonstrated that hydroxyurea generates the nitric oxide (NO) radical in vivo, and we therefore hypothesized that NO-donor properties might determine the hemoglobin phenotype. We treated both K562 erythroleukemic cells and human erythroid progenitor cells with S-nitrosocysteine (CysNO), an NO donor, and found similar dose- and time-dependent induction of gamma-globin mRNA and HbF protein as we observed with hydroxyurea. Both hydroxyurea and CysNO increased cGMP levels, and the guanylyl cyclase inhibitors ODQ, NS 2028, and LY 83,538 abolished both the hydroxyurea- and CysNO-induced gamma-globin expression. These data provide strong evidence for an NO-derived mechanism for HbF induction by hydroxyurea and suggest possibilities for therapies based on NO-releasing or -potentiating agents.

Erythropoietin and hypoxia increase erythropoietin receptor and nitric oxide levels in lung microvascular endothelial cells

Acute lung exposure to low oxygen results in pulmonary vasoconstriction and redistribution of blood flow. We used human microvascular endothelial cells from lung (HMVEC-L) to study the acute response to oxygen stress. We observed that hypoxia and erythropoietin (EPO) increased erythropoietin receptor (EPOR) gene expression and protein level in HMVEC-L. In addition, EPO dose- and time-dependently stimulated nitric oxide (NO) production. This NO stimulation was evident despite hypoxia induced reduction of endothelial NO synthase (eNOS) gene expression. Western blot of phospho-eNOS (serine1177) and eNOS and was significantly induced by hypoxia but not after EPO treatment. However, iNOS increased at hypoxia and with EPO stimulation compared to normal oxygen tension. In accordance with our previous results of NO induction by EPO at low oxygen tension in human umbilical vein endothelial cells and bone marrow endothelial cells, these results provide further evidence in HMVEC-L for EPO regulation of NO production to modify the effects of hypoxia and cause compensatory vasoconstriction.

Assessment of bone marrow microvessel density in chronic lymphocytic leukemia.

IntroductionAngiogenesis is a physiologic process of new blood vessels formation mediated by various cytokines called proangiogenic and antiangiogenic factors. Enhancement of angiogenesis in chronic lymphocytic leukemia (CLL) has been recognized more recently. Our study assesses CD34 and von Willebrand factor (vWf) expression and microvessel density (MVD) in the bone marrow of patients with CLL. Aims(1) To assess bone marrow MVD in CLL using 2 different monoclonal antibodies, CD34 and vWf; and (2) To examine the possible association of marrow MVD and clinical course, pattern of marrow infiltration, Rai stage, CD38 positivity, and cytogenetic abnormalities detected by fluorescence in situ hybridization. Materials and MethodsBone marrow specimens from 33 patients with CLL and 10 controls were studied. A single microvessel was defined as any vessel with a clear lumen. The screening of the slides was carried out by hotspot method. The slides were initially screened at low power to identify the areas with highest number of microvessel or vascularity hotspot. The count of microvessel in a sufficiently extended field (40× objective lens, 10× ocular lens) was then performed. The mean value of 10 most vascularized areas at 400× field was considered as MVD for a sample. ResultsThere was a significant difference between MVD counts according to the antibody used. MVD was higher using CD34 versus vWF (CD34: mean ± SD, 35.91±15.7; 95% confidence interval of mean, 30.34-41.48 vessels/field versus vWF: 8.15±4.65; 95% confidence interval of mean, 4.11-12.44 vessels/field; P<0.0001]. Bone marrow MVD detected by CD34 was significantly higher in patients with CD38 expression more than 30% (P=0.006) and in patients with unfavorable cytogenetic abnormalities. However, no significant MVD differences were detected between CLL subgroups with regard to clinical course, pattern of marrow infiltration, and Rai stage. Bone marrow MVD in patients with CLL was significantly higher than that in controls (P<0.0001). ConclusionsMVD assessment using anti-CD34 resulted in higher MVD counts than when using anti-vWF antibody. However, no MVD differences were detected between CLL subgroups subdivided according to the above-mentioned prognostic factors except CD38 expression and genetic abnormalities.

Proliferation and differentiation markers of colorectal adenocarcinomaand 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.

Nitric Oxide / Cyclic Nucleotide Regulation of Globin Genes in Erythropoiesis

Vladan P. Cokic1, Bojana B. Beleslin-Cokic2, Gordana Jovcic1, Raj K. Puri3 and Alan N. Schechter4 1Laboratory of Experimental Hematology, Institute for Medical Research, Belgrade, 2Clinic of Endocrinology, Diabetes and Diseases of Metabolism, School of Medicine, University Clinical Center, Belgrade, 3Division of Cellular and Gene Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, 4Molecular Medicine Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 1,2Serbia 3,4USA