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ABSTRACT: Nitric oxide (NO) is an endogenous vasodilator molecule synthetized from L-arginine by a family of nitric oxide synthases. In differentiated human endothelial cells, it is well known that L-arginine uptake via cationic amino acid transporters (y/CAT) or system yL is required for the NO synthesis via endothelial nitric oxide synthase, but there are no reports in human endothelial progenitor cell (hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC-14d) to characterize the L-arginine transport and NO synthesis in those cells. L-arginine transport and NO synthesis were analyzed in the presence or absence of N-ethylmaleimide or L-nitroarginine methyl ester, as inhibitors of y/CAT system and nitric oxide synthases, respectively. The results showed that L-arginine uptake is higher in hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least 2 transporter systems in hEPC: a high affinity transporter system (Km= 4.8 ± 1.1 μM for hEPC-3d and 6.1 ± 2.4 μM for hEPC-14d) and a medium affinity transporter system (Km = 85.1 ± 4.0 μM for hEPC-3d and 95.1 ± 8 μM for hEPC-14d). Accordingly, hEPC expressed mRNA and protein for CAT-1 (ie, system y) and mRNA for 2 subunits of yL system, yLAT1, and 4F2hc. Higher L-citruline production and NO bioavailability (4-fold), and endothelial nitric oxide synthase expression (both mRNA and protein) were observed in hEPC-14d compared with hEPC-3d. Finally, the high L-citruline formation observed in hEPC-14d was blocked by N-ethylmaleimide. In conclusion, this study allowed to identity a functional L-arginine/NO pathway in two hEPC differentiation stages, which improves the understanding of the physiology of these precursor cells.
Journal of cardiovascular pharmacology 11/2012; 60(5):439-49. · 2.83 Impact Factor
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ABSTRACT: ABTRACT: Nitric oxide (NO) is an endogenous vasodilator molecule synthetized from L-arginine by a family of nitric oxide synthases (NOS). In differentiated human endothelial cells, it is well known that L-arginine uptake via cationic amino acid transporters (y/CAT) or system yL is required for the NO synthesis via endothelial NOS (eNOS), but there are no reports in human endothelial progenitor cell (hEPC). Therefore, we isolated hEPCs from peripheral blood of healthy donors and cultured them for either 3 (hEPC-3d) or 14 days (hEPC-14d) in order to characterize the L-arginine transport and NO synthesis in those cells. L-arginine transport and NO synthesis were analyzed in the presence or absence of N-ethylmaleimide (NEM) or L-nitroarginine methyl ester (L-NAME), as inhibitors of y/CAT system and NOS, respectively. The results showed that L-arginine uptake is higher in hEPC-14d than in hEPC-3d. Kinetic parameters for L-arginine transport showed the existence of at least two transporter systems in hEPC: a high affinity transporter system (Km= 4.8±1.1 μM for hEPC-3d and 6.1±2.4 μM for hEPC-14d) and a medium affinity transporter system (Km= 85.1±4.0 μM for hEPC-3d and 95.1±8 μM for hEPC-14d). Accordingly, hEPC expressed mRNA and protein for CAT-1 (i.e., system y) and mRNA for two subunits of yL system, yLAT1 and 4F2hc. Higher L-citruline production and NO bioavailability (4-fold), as well as eNOS expression (both mRNA and protein) were observed in hEPC-14d compared to hEPC-3d. Finally, the high L-citruline formation observed in hEPC-14d was blocked by NEM. In conclusion, this study allowed to identity a functional L-arginine/NO pathway in two hEPC differentiation stages, which improves the understanding of the physiology of these precursor cells.
Journal of cardiovascular pharmacology 07/2012; · 2.83 Impact Factor
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ABSTRACT: Human endothelial progenitor cells (hEPC) are recruited to sites of neovascularization where they differentiate into endothelial cells. The signals/factors responsible for hEPC migration and adhesion to sites of injury are not well understood. Elevated levels of adenosine are known to increase mature endothelial cell migration in response to tissue injury. However, the understanding of the role of adenosine in the physiology of hEPC is very limited. Using quantitative polymerase chain reaction and western blot analyses, we detected the expression of the adenosine receptors A₂A, A₂B, and A₃ in hEPC. Stimulation of adenosine receptors using adenosine or the nonselective agonist adenosine-5'-N-ethylcarboxamide (NECA) increased hEPC migration in 1.4-fold and 2.1-fold (P < 0.01), respectively. Stimulation of hEPC using the A₂A-specific agonist CGS-21680 resembled the effect observed in migration when using adenosine or NECA. Consequently, NECA and CGS-21680-stimulated migration of hEPC were reverted using the A₂A receptor antagonist ZM-241385. NECA-stimulated migration was inhibited in dose-dependent manner using MRS-1523 (Ki of 147 ± 0.016 nM), MRS-1754 (Ki of 1900 ± 0.02 nM), or ZM-241385 (Ki of 0.2 ± 0.01 nM). In conclusion, adenosine stimulates hEPC migration by activating A₂A and A₃ but not A₂B receptors and provides evidence to support a role of adenosine in modulating angiogenic capacity of hEPC.
Journal of cardiovascular pharmacology 01/2012; 59(5):397-404. · 2.83 Impact Factor
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Andrea Searle,
Leonardo Gómez-Rosso,
Tomás Meroño,
Carlos Salomon,
Daniel Durán-Sandoval,
Gustavo Giunta,
Carlos Grant,
Carlos Calvo,
Liliana Lamperti,
Fernando Brites, Claudio Aguayo
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ABSTRACT: To evaluate in vitro the effects of serum and LDL fractions isolated from hypercholesterolemic patients on nitric oxide (NO) synthesis and reactive oxygen species (ROS) production by human umbilical vein endothelial cells (HUVECs).
Serum and LDL isolated from subjects with high (n=18) and normal (n=21) LDL-cholesterol levels were analyzed on NO synthesis and ROS production in vitro models of HUVECs. LDL was furthers characterized in their chemical composition and activities of lipoprotein-associated phospholipase A(2) (Lp-PLA(2)), cholesteryl ester transfer protein (CETP) and paraoxonase.
NO bioavailability was significantly lower and ROS production higher in HUVECs incubated with serum samples from patients with high LDL-cholesterol levels in comparison to control subjects. Moreover, hypercholesterolemic patients presented higher CETP and Lp-PLA(2) activities than control subjects. LDL fractions isolated from patients and controls were not different in their chemical composition, Lp-PLA(2) activity, and their capacity to reduce NO synthesis and increase ROS production.
Alterations of serum from hypercholesterolemic patients could be due to the increment in LDL concentration, main Lp-PLA(2) carrier, and not to LDL composition or intrinsic Lp-PLA(2) activity.
Clinical biochemistry 10/2010; 44(2-3):171-7. · 2.02 Impact Factor
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ABSTRACT: High D-glucose reduces human equilibrative nucleoside transporter 1 (hENT1)-mediated adenosine uptake involving endothelial nitric oxide synthase (eNOS), mitogen-activated protein (MAP) kinase kinases 1 and 2/MAP kinases p42/44 (MEK/ERKs), and protein kinase C (PKC) activation in human umbilical vein endothelium (HUVEC). Since NO represses SLC29A1 gene (hENT1) promoter activity we studied whether D-glucose-reduced hENT1-adenosine transport results from lower SLC29A1 expression in HUVEC primary cultures. HUVEC incubation (24 h) with high D-glucose (25 mM) reduced hENT1-adenosine transport and pGL3-hENT1(-1114) construct SLC29A1 reporter activity compared with normal D-glucose (5 mM). High D-glucose also reduced pGL3-hENT1(-1114) reporter activity compared with cells transfected with pGL3-hENT1(-795) construct. N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), PD-98059 (MEK1/2 inhibitor), and/or calphostin C (PKC inhibitor) blocked D-glucose effects. Insulin (1 nM) and phorbol 12-myristate 13-acetate (PMA, 100 nM, PKC activator), but not 4alpha-phorbol 12,13-didecanoate (4alphaPDD, 100 nM, PMA less active analogue) reduced hENT1-adenosine transport. L-NAME and PD-98059 blocked insulin effects. L-NAME, PD-98059, and calphostin C increased hENT1 expression without altering protein or mRNA stability. High D-glucose increased Sp1 transcription factor protein abundance and binding to SLC29A1 promoter, phenomena blocked by L-NAME, PD-98059, and calphostin C. Sp1 overexpression reduced SLC29A1 promoter activity in normal D-glucose, an effect reversed by L-NAME and further reduced by S-nitroso-N-acetyl-L,D-penicillamine (SNAP, NO donor) in high D-glucose. Thus, reduced hENT1-mediated adenosine transport in high D-glucose may result from increased Sp1 binding to SLC29A1 promoter down-regulating hENT1 expression. This phenomenon depends on eNOS, MEK/ERKs, and PKC activity, suggesting potential roles for these molecules in hyperglycemia-associated endothelial dysfunction.
Journal of Cellular Physiology 07/2008; 215(3):645-56. · 3.87 Impact Factor
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The Journal of Physiology 08/2004; 529(3):777 - 790. · 4.72 Impact Factor
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ABSTRACT: D-glucose infusion and gestational diabetes induce vasodilatation in humans and increase L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells. High D-glucose (25 mmol/L, 2 minutes) induced membrane hyperpolarization and an increase of L-arginine transport (V(max) 6.1+/-0.7 versus 4.4+/-0.1 pmol/ microg protein per minute) with no change in transport affinity (K(m) 105+/-9 versus 111+/-16 micromol/L). L-[3H]citrulline formation and intracellular cGMP, but not intracellular Ca2+, were increased by high D-glucose. The effects of D-glucose were mimicked by levcromakalim (ATP-sensitive K+ channel blocker), paralleled by p42/p44(mapk) and Ser(1177)-endothelial NO synthase phosphorylation, inhibited by N(G)-nitro-L-arginine methyl ester (L-NAME; NO synthesis inhibitor), glibenclamide (ATP-sensitive K+ channel blocker), KT-5823 (protein kinase G inhibitor), PD-98059 (mitogen-activated protein kinase kinase 1/2 inhibitor), and wortmannin (phosphatidylinositol 3-kinase inhibitor), but they were unaffected by calphostin C (protein kinase C inhibitor). Elevated D-glucose did not alter superoxide dismutase activity. Our findings demonstrate that the human fetal endothelial L-arginine/NO signaling pathway is rapidly activated by elevated D-glucose via NO and p42/44(mapk). This could be determinant in pathologies in which rapid fluctuations of plasma D-glucose may occur and may underlie the reported vasodilatation in early stages of diabetes mellitus.
Circulation Research 02/2003; 92(1):64-72. · 9.49 Impact Factor
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ABSTRACT: Chronic incubation with elevated D-glucose reduces adenosine transport in endothelial cells. In this study, exposure of human umbilical vein endothelial cells to 25 mmol/L D-glucose or 100 micromol/L ATP, ATP-gamma-S, or UTP, but not ADP or alpha,beta-methylene ATP, reduced adenosine transport with no change in transport affinity. Inhibition of transport by D-glucose, ATP, and ATP-gamma-S was associated with reduced maximal binding, with no changes in the apparent dissociation constant for nitrobenzylthioinosine (NBMPR). A significant reduction (approximately 60+/-10%, P<0.05; n=6) in the number of human equilibrative NBMPR-sensitive nucleoside transporters (hENT1s) per cell (1.8+/-0.1x10(6) in 5 mmol/L D-glucose) and in hENT1 mRNA levels was observed in cells exposed to D-glucose or ATP-gamma-S. Incubation with elevated D-glucose, but not with D-mannitol, increased the ATP release by 3+/-0.2-fold. The effects of D-glucose and nucleotides on the number and activity of hENT1 and hENT1 mRNA were blocked by reactive blue 2 (nonspecific P2Y purinoceptor antagonist), suramin (Galpha(s) protein inhibitor), or hexokinase but not by pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (nonselective P2 purinoceptor antagonist). Our findings demonstrate that inhibition of adenosine transport via hENT1 in endothelial cells cultured in 25 mmol/L D-glucose could be due to stimulation of P2Y2 purinoceptors by ATP, which is released from these cells in response to D-glucose. This could be a mechanism to explain in part the vasodilatation observed in the early stages of diabetes mellitus or in response to D-glucose infusion.
Circulation Research 03/2002; 90(5):570-7. · 9.49 Impact Factor
