1. Hydroxyurea induces the eNOS-cGMP pathway in endothelial cells
by Vladan P. Cokic, Bojana B. Beleslin-Cokic, Melanija Tomic, Stanko S. Stojilkovic, Constance T. Noguchi, and Alan N. Schechter
Blood
Volume 108(1):
July 1, 2006
©2006 by American Society of Hematology

2. Measurement of NO production in endothelial cells during incubation with hydroxyurea and 2 other cytostatic agents.
Measurement of NO production in endothelial cells during incubation with hydroxyurea and 2 other cytostatic agents. (A) HUVECs were treated with different concentrations of hydroxyurea (10 and 20 μM). (B) TrHBMECs, as well as HUVECs, were treated with different concentrations of hydroxyurea. (C) HUVECs were treated with different concentrations of butyric acid sodium salt (0.5 and 1 mM) and 5-azacytidine (10 and 20 μM). (D) TrHBMECs were treated with the same concentrations of butyric acid sodium salt and 5-azacytidine as HUVECs. Results are from 3 to 4 independent experiments: HUVECs are from different cryopreserved cells pooled from several donors, whereas TrHBMECs are thawed from a new cryovial for each experiment. Values are mean ± SEM (n = 3).
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology

3. Measurement of NO levels during inhibition of eNOS in endothelial cells incubated with hydroxyurea (HU).
Measurement of NO levels during inhibition of eNOS in endothelial cells incubated with hydroxyurea (HU). (A) Hydroxyurea (20 μM) induction of NO is inhibited by competitive eNOS inhibitor NG-nitro-l-arginine (0.1 mM) in HUVECs in the presence of supplementary 0.1 mM l-arginine (*P < .05 versus cells treated with hydroxyurea and NG-nitro-l-arginine). (B) L-NAME (0.1 mM) also inhibited hydroxyurea (20 μM) induction of NO production in TrHBMECs in the presence of supplementary 0.1 mM l-arginine (*P < .05 versus cells treated with hydroxyurea and L-NAME). Values represent mean ± SEM (n = 3).
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology

4. Measurement of NO production in endothelial cells during incubation with hydroxyurea in presence or not of inhibitors of PKA and PKB/Akt.
Measurement of NO production in endothelial cells during incubation with hydroxyurea in presence or not of inhibitors of PKA and PKB/Akt. (A) After preincubation period with 20 μM H-89 (PKA inhibitor), 50 μM LY294002, and 0.1 μM wortmannin (PI 3-kinase inhibitors), HUVECs were treated with 2 different concentrations of hydroxyurea (20 and 50 μM), and aliquots were collected for measurement after 1 hour of incubation. (B) TrHBMECs were preincubated with PKA and PI 3-kinase inhibitors, and measurements were made after 1 hour of incubation with hydroxyurea. Values are mean ± SEM (n = 3). *P < .05 and **P < .01 compared with cells treated with only hydroxyurea.
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology

5. Hydroxyurea phosphorylated eNOS in HUVECs
Hydroxyurea phosphorylated eNOS in HUVECs. (A) Left panel: hydroxyurea (at concentrations of 30 and 50 μM) induced phosphorylation of eNOS at Ser1177 after 30 seconds of treatment and the signal remained for more than 1 minute; bradykinin (1 μM) induced sim...
Hydroxyurea phosphorylated eNOS in HUVECs. (A) Left panel: hydroxyurea (at concentrations of 30 and 50 μM) induced phosphorylation of eNOS at Ser1177 after 30 seconds of treatment and the signal remained for more than 1 minute; bradykinin (1 μM) induced similar phosphorylation. Right panel: corresponding total eNOS protein levels in HUVECs during incubation with hydroxyurea and bradykinin. Results are representative of 4 independent experiments. (B) Densitometric analysis of panel A during 5 minutes of incubation of HUVECs with hydroxyurea and bradykinin. Results are represented as the ratio of pSer1177 to total eNOS bands intensity. (C) PI 3-kinase inhibitors: LY (LY, 50 μM) and wortmannin (W, 100 nM) demonstrated incomplete inhibition of eNOS phosphorylation at Ser1177 in HUVECs after 1 minute of incubation with hydroxyurea (50 μM) (upper bands). PKA inhibitor H-89 (20 μM) induced complete inhibition of eNOS phosphorylation at Ser1177 in HUVECs after 1 minute of incubation with hydroxyurea (50 μM) (upper band). Lower bands show total eNOS protein levels. Results are representative of 4 independent experiments.
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology

6. Hydroxyurea increased cAMP and cGMP levels in HUVECs
Hydroxyurea increased cAMP and cGMP levels in HUVECs. (A) Hydroxyurea showed dose-dependent induction of cAMP levels in HUVECs after 30 minutes of incubation.
Hydroxyurea increased cAMP and cGMP levels in HUVECs. (A) Hydroxyurea showed dose-dependent induction of cAMP levels in HUVECs after 30 minutes of incubation. (B) Hydroxyurea showed dose-dependent induction of cGMP levels in HUVECs after 30 minutes of incubation, whereas pretreatment with L-NAME inhibited this effect. Values represent mean ± SEM (n = 3); **P < .01 compared with cells untreated with hydroxyurea.
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology

7. Effects of hydroxyurea on intracellular calcium concentration in HUVECs. (A) The effect of hydroxyurea on [Ca2+]i was independent of the time of its application (left panel vs right panel) and was not mimicked by the addition of medium 199 (right panel).
Effects of hydroxyurea on intracellular calcium concentration in HUVECs. (A) The effect of hydroxyurea on [Ca2+]i was independent of the time of its application (left panel vs right panel) and was not mimicked by the addition of medium 199 (right panel). Traces shown are representative from at least 3 experiments, each performed on 15 to 30 single cells during incubation with hydroxyurea. Gray areas indicate the duration of hydroxyurea treatment. Notice a variety in times of initiation of [Ca2+]i response after the addition of hydroxyurea. (B) Dependence of hydroxyurea-induced calcium response on calcium mobilization from intracellular stores. Comparison of [Ca2+]i signals induced by hydroxyurea (bottom trace) and endothelin-1, the native calcium-mobilizing agonist for these cells (upper panel). (C) Ionomycin-induced [Ca2+]i signals in controls (upper trace) and hydroxyurea-treated cells (bottom trace). (D) The lack of effects of hydroxyurea on [Ca2+]i response in cells with blocked endoplasmic reticulum calcium pump with thapsigargin. To avoid the UV damage of cells, the ratio of light intensities during thapsigargin treatment was followed at a rate of 1 point per 20 seconds.
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology

8. Hydroxyurea effects on blood vessels.
Hydroxyurea effects on blood vessels. Hydroxyurea oxidizes oxy- and deoxy-hemoglobin to methemoglobin (metHb), which may react directly with other molecules of hydroxurea (or via hydroxylamine) to form nitrosylhemoglobin (HbNO), which can slowly release NO. Hydroxyurea may also chemically or enzymatically (peroxidase, urease, catalase) decompose to NO, as well as directly effect intracellular levels of cations, production of VCAM-1, and red-cell adhesion, among others. Our data show that hydroxyurea also stimulates the phosphorylation and thus activation of endothelial NOS (eNOS) with resultant production of NO. This effect is modulated by increased intracellular calcium levels and increases in cAMP, which stimulates phosphorylation at Ser1177 by PKA. NO production is regulated by both PKA and PKB/Akt. The increase in eNOS activity presumably leads to increases in cGMP in endothelial and surrounding cells; this increase results in many of the pleiotropic effects of endothelial-produced NO. SM indicates smooth muscle cells; E, endothelial cells.
Vladan P. Cokic et al. Blood 2006;108:
©2006 by American Society of Hematology