Placenta
Volume 31, Issue 10 , Pages 928-936, October 2010

Differential expression of functional nucleoside transporters in non-differentiated and differentiated human endothelial progenitor cells

  • E. Guzmán-Gutiérrez

      Affiliations

    • Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, Universidad de Concepción, Chile
    • Actual address: Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile.
    • ,
  • C. Sandoval

      Affiliations

    • Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, Universidad de Concepción, Chile
    • ,
  • E. Nova

      Affiliations

    • Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, Universidad de Concepción, Chile
    • ,
  • J.L. Castillo

      Affiliations

    • Department of Medical Specialties, Faculty of Medicine, Universidad de Concepción, Chile
    • ,
  • J.C. Vera

      Affiliations

    • Department of Physiopathology, Faculty of Biological Sciences, Universidad de Concepción, Chile
    • ,
  • L. Lamperti

      Affiliations

    • Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, Universidad de Concepción, Chile
    • ,
  • B. Krause

      Affiliations

    • Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile
    • ,
  • C. Salomón

      Affiliations

    • Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile
    • ,
  • C. Sepúlveda

      Affiliations

    • Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile
    • ,
  • C. Aguayo

      Affiliations

    • Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, Universidad de Concepción, Chile
    • These authors contributed equally to this study.
    • ,
  • L. Sobrevia

      Affiliations

    • Cellular and Molecular Physiology Laboratory (CMPL) & Perinatology Research Laboratory (PRL), Division of Obstetrics and Gynaecology, Medical Research Centre (CIM), School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, P.O. Box 114-D, Santiago, Chile
    • Corresponding Author InformationCorresponding author. Tel.: +56 2 3548118; fax: +56 2 6321924.
    • These authors contributed equally to this study.

Accepted 29 July 2010. published online 23 August 2010.

Article Outline

Abstract 

Extracellular adenosine removal is via human equilibrative nucleoside transporters 1 (hENT1) and 2 (hENT2) in the endothelium, thus regulating adenosine-induced revascularization and angiogenesis. Since human endothelial progenitor cells (hEPCs) promote revascularization, we hypothesize differential expression of nucleoside transporters in hEPCs. hEPCs were cultured 3 (hEPC-3d) or 14 (hEPC-14d) days. RT-PCR for prominin 1, CD34, octamer-4, kinase insert domain receptor, oxidized low-density lipoprotein (lectin-like) receptor 1 and tyrosine endothelial kinase was used to evaluate phenotypic differentiation. Flow cytometry was used to estimate CD34+/KDR− (non-differentiated), CD34−/KDR+ (differentiated) or CD34+/KDR+ (mixed) cell populations. Adenosine transport was measured in absence or presence of sodium, S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1–10 μM), inosine, hypoxanthine or guanine (0.1–5 mM), hENTs protein abundance by western blot, and hENTs, hCNT1, hCNT2 and hCNT3 mRNA expression by real time RT-PCR. hEPC-3d cells were CD34+/KDR− compared with hEPC-14d cells that were CD34−/KDR+. hEPC-3d cells exhibit hENT1-like adenosine transport (NBTI-sensitive, Na+-independent), which is absent in hEPC-14d cells. hEPC-14d cells exhibit two transport components: component 1 (NBTI insensitive, Na+-independent) and component 2 (NBTI insensitive, Na+-dependent, Hill coefficient ∼1.8), the latter resembling CNT3-like transport. hEPC-3d cells express hENT1 protein and mRNA, which is reduced (∼90%) in hEPC-14d cells, but instead only hCNT3 mRNA is expressed in this cell type. hENT2, hCNT1 and hCNT2 were undetectable in hEPCs. Thus, hEPCs exhibit a differential expression of hENT1 and hCNT3 functional nucleoside transporters, which could be related with its differentiation stage.

Keywords: Endothelial progenitor cells, Adenosine, Transport, Differentiation

 

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1. Introduction 

Human endothelial progenitor cells (hEPCs) from peripheral blood of adult subjects are involved in re-endothelialization and neovascularization of injured and ischemic tissues [1], [2] as well as in regeneration of endothelial cells in the utero-placental circulation in pre-eclampsia [3]. Interestingly, the endogenous nucleoside adenosine is also involved in promoting neovascularization and angiogenesis in human tissues [4], [5], [6], [7], [8], a phenomenon increased when adenosine uptake is reduced in endothelium [6], [7], [8]. Thus, a crucial role for nucleoside membrane transporters in adenosine vascular effects has been proposed [6], [7]. However, nothing is reported regarding the role of nucleoside transporters in the biological functions of hEPCs [2], [9].

Nucleoside transport is mediated by at least two families of proteins in mammalian cells, i.e., equilibrative, Na+-independent (ENTs) and concentrative, Na+-dependent (CNTs) nucleoside transporters [10], [11]. Human endothelium expresses functional isoforms 1 (hENT1) and 2 (hENT2) under physiological conditions, but no reports on CNTs expression and/or transport activity are available [6], [11]. Since inhibition of nucleoside uptake reduces endothelial cells differentiation [12], and inhibition of the nucleoside transporters-mediated uptake of the nucleoside-type antitumoral drug gemcitabine induces hEPCs mobilization [13], [14], and adenosine induces EPCs differentiation into endothelial cells [15], altered removal of nucleosides has a consequence modulating endothelium and EPCs biological function.

Other studies show that adhesion of murine embryonic EPCs (eEPCs) to cardiac microvascular endothelial cells and its retention in isolated mouse hearts are increased by adenosine requiring activation of A1 adenosine receptors [16]. These findings strongly suggest that adenosine acting on adenosine receptors in eEPCs plays a role in modulating their biological functions. In addition, A2A adenosine receptors seem essential in promoting mouse bone marrow-derived mesenchymal stem cell differentiation [17]; however, nothing is reported regarding the potential effects of adenosine on hEPCs differentiation [2], [9]. We speculate that adenosine transport could be a phenomenon involved in hEPCs differentiation. Our results show that hEPCs exhibit nucleoside transport activity and that non-differentiated hEPCs express hENT1, but not hENT2 or hCNT3; however, differentiation of hEPCs in culture is associated with lost of hENT1- and expression of hCNT3-like transport activity. A potential differential expression of hENTs and hCNTs is a phenomenon that could alter the biological functions of hEPCs, perhaps playing a role in their differentiation.

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2. Materials and methods 

2.1. Human endothelial progenitor cells isolation 

Human endothelial progenitor cells (hEPCs) were isolated as previously described [18], [19], [20]. The total mononuclear cells fraction was isolated from peripheral venous blood from healthy volunteers by Ficoll-Histopaque (Sigma Chemical Co, St Louis, MO, USA) gradient separation. Before they were used for cell culture, the mononuclear cells fraction was washed (×3) by centrifugation (250g, 10 min) with phosphate buffer saline [PBS (mM): 137 NaCl, 2.7 KCl, 1.8 KH2PO4, 8.2 Na2HPO4, pH 7.4, 37 °C] solution as described [18], [19], [20]. This study conforms to the principles outlined in the Declaration of Helsinki. Ethic committee approval and written consent of donors were obtained.

2.2. Human endothelial progenitor cells culture 

hEPCs were plated (1.5 × 106 cells/cm2) on fibronectin-coated culture slides (Sigma) and cultured (37 °C, 5% CO2) up to passage 1 in primary culture medium (PCM) composed of endothelial growth medium (GIBCO BRL Life Technologies, Bethesda, MD, USA) [18], [19], [20] containing 5 mM d-glucose, 15% foetal calf serum, 10 μg/mL of human vascular endothelial growth factor A and 100 U/mL penicillin–streptomycin (GIBCO) [18], [19], [20]. PCM was changed every 3 days. Prior to experiments (12 h) cells were exposed to PCM containing 2% of sera.

2.3. Human endothelial progenitor cells phenotype characterization 

Since hEPCs differentiate to endothelium at ∼14 days [18], [19], [20], hEPCs phenotypic differentiation was evaluated in cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d). Transcripts for prominin 1 (CD133), CD34, octamer-4 (Oct-4), tyrosine endothelial kinase (Tie-2), kinase insert domain receptor (KDR) and oxidized low-density lipoprotein (lectin-like) receptor 1 (Lox-1) were determined by reverse transcription-polymerase chain reaction (RT-PCR).

2.4. Flow cytometry 

Flow cytometry was performed on an argon laser FACSCalibur (BD Biosciences, Pharmingen, San Diego, CA, USA) with an excitation wavelength at 488 nm. Detection of CD34+/KDR−, CD34−/KDR+ or CD34+/KDR+ cell populations was performed in hEPC incubated (20 min, 22 °C) with monoclonal antibodies against human phycoerytrin conjugated CD34 (Becton Dickinson, San Diego, CA, USA) and CarboxyFluorescein conjugated KDR (R&D System Inc., Minneapolis, MN, USA). Following this incubation period, 50.000 events were analyzed per sample and cell count was presented as a percentage of total number of cells in the sample [19], [20].

2.5. Adenosine transport 

Overall adenosine transport (0–30 μM adenosine, 85 nM [3H]adenosine, 2 μCi/mL, 20 s, 22 °C) was measured as described [21]. Briefly, cells were exposed (30 min before transport assays) to Krebs solution [(mM): NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1, Hepes 20, CaCl2 2.5, MgCl2 1 (pH 7.4)] without or with S-(4-nitrobenzyl)-6-thio-inosine (NBTI, 1 μM, an inhibitory concentration for hENT1 transport activity) (Sigma Chemical Co, Atlanta, GA, USA) [6], [10], [21]. The difference between total adenosine transport and transport in the presence of 1 μM NBTI was defined as ENT1 (NBTI-sensitive)-mediated adenosine transport (i.e., hereafter referred as hENT1-adenosine transport) [21]. In some experiments sodium (Na+) in the transport medium was replaced by equimolar concentration of N-methylglucamine-HCl (NMG).

Overall adenosine transport at initial rates was adjusted to the Michaelis–Menten hyperbola plus a non-saturable, linear component as described [7]:

where v is adenosine uptake, Vmax is maximal velocity of transport, Km is Michaelis–Menten parameter, [Ado] is adenosine concentration and m is slope of the linear component of transport. The non-saturable, linear component in the range of adenosine concentrations used in this study is defined by m·[Ado] [7].

Saturable adenosine transport kinetic parameters Vmax and Km were calculated from data where the non-saturable, linear uptake of adenosine was substracted from overall transport data and fitted to the Michaelis–Menten hyperbola assuming a single saturable transport system for adenosine for hEPC-3d cells:

In hEPC-14d cells total adenosine transport was semi-saturable and fitted best by a double Michaelis–Menten equation, i.e., for two saturable transport systems (hereafter referred as component 1 and component 2) acting in parallel. The relative contribution of the components 1 and 2 in hEPC-14d cells on total adenosine transport in the presence of Na+ (C1/C2-NaF) or NMG (C1/C2-NMGF) was estimated from the maximal transport capacity (Vmax/Km) values for total adenosine transport by:

where x is Na+ or NMG, C1Vmax, C1Km, C2Vmax and C2Km are kinetic parameters for adenosine transport via component 1 and 2 in the presence of Na+ or NMG, respectively [6], [7]. In experiments where Na+ (Na) was replaced in the medium by NMG (NMG), the relative contribution of this ion to adenosine transport was estimated by 1/(Na/NMG)xFy where x is either hEPC-3d or hEPC-14d cells and y is either the unique transport component in hEPC-3d cells (i.e., 1/(Na/NMG)3dF), or component 1 (1/(Na/NMG)14dFC1) or 2 (1/(Na/NMG)14dFC2) for hEPC-14d cells.

hENT1-adenosine (10 μM) transport was also measured in absence or presence of the nucleoside inosine (0.1–1 mM), or the nucleobases guanine (1–5 mM) or hypoxanthine (1–5 mM). The apparent inhibition constants (Ki) of hENT1-adenosine transport by these molecules were calculated by the tight-inhibition Morrison equation:

where Km is the apparent Km value for adenosine transport in hEPC-3d or hEPC-14d cells, [Ado] is 10 μM and IC50 is the half-maximal inhibitory concentration of the inhibitors [22]. Adenosine transport (10 μM) in hEPC-14d was also assayed in Krebs containing increasing concentrations of Na+ (0–100 mM) from where Hill coefficient (n) was calculated from Hill equation.

2.6. Western blotting 

Proteins (70 μg) separated by polyacrylamide gel (10%) electrophoresis were transferred to Immobilon-P polyvinylidene difluoride membranes (BioRad Laboratories, Hertfordshire, UK) and probed with primary polyclonal rabbit anti-hENT1 (1:1000), anti-hENT2 (1:250) or anti-β-actin (1:2000)(Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies as described [21]. Membranes were rinsed in Tris buffer saline Tween, and incubated (1 h) in TBS-T/0.2% BSA containing horseradish peroxidase-conjugated goat anti-rabbit antibody. Proteins were detected by enhanced chemiluminescence (film exposure time was 5 min) and quantitated by densitometry as described [21].

2.7. Isolation of total RNA and reverse transcription 

Total RNA was isolated using the Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNA quality and integrity were insured by gel visualization and spectrophotometric analysis (OD260/280), quantified at 260 nm and precipitated to obtain 4 μg/μL. Aliquots (1 μg) of total RNA were reversed-transcribed into cDNA using oligo(dT18) plus random hexamers (10-mer) primers and avian Moloney murine leukaemia virus reverse transcriptase (MMLV-RT)(Invitrogen) for 1 h at 37 °C as described [21].

2.8. RT-PCR 

Semi-quantitative RT-PCR was performed in 20 μL containing 2 μL 10× PCR buffer, 0.6 μL 50 mM Mg+2, 0.8 μL dNTP’s, 13.2 μL RNAase-free H2O, 0.4 μL Taq DNA polymerase (Invitrogen) and sequence-specific oligonucleotide primers for human ENT1, ENT2, CNT1, CNT2, CNT3, CD133, CD34, Oct-4, Tie-2, KDR and Lox-1 (0.5 μM). For hENT1 and hENT2 transcripts, samples were incubated 4 min at 95 °C, followed by 25 cycles of 30 s at 95 °C, 30 s at 57 °C, 30 s at 72 °C, and 7 min final extension at 72 °C. For hCNT1, hCNT2 and hCNT3 transcripts, samples were incubated 1 min at 95 °C, followed by 35 cycles of 60 s at 95 °C, 60 s at 60 °C, 60 s at 72 °C, and 10 min final extension at 72 °C. For CD133, CD34, Oct-4, Tie-2, KDR and Lox-1, samples were incubated 10 min at 95 °C, followed by 35 cycles of 30 s at 95 °C, 30 s at 57 °C for CD133, 60 °C for CD34 and Oct-4, or 62 °C for Tie-2, KDR and Lox-1, 30 s at 72 °C, and 7 min final extension at 72 °C. β-Actin mRNA was internal reference [21]. RT-PCR products were sequenced as described [21]. Oligonucleotide primers are given in Table 1. Expected size products for hENT1 (151 bp), hENT2 (232 bp), hCNT1 (201 bp), hCNT2 (255 bp), hCNT3 (69 bp), CD133 (240 bp), CD34 (380 bp), Oct-4 (169 bp), Tie-2 (490 bp), KDR (490 bp), Lox-1 (193 bp) and β-actin (350 bp) were confirmed in agarose gel separation electrophoresis (1.5%).

Table 1. Oligonucleotide primers for RT-PCR.
hENT1(sense)5′-TCTCCAACTCTCAGCCCACCAA-3′
hENT1(anti-sense)5′-CCTGCGATGCTGGACTTGACCT-3′
hENT2(sense)5′-TCTCCAACTCTCAGCCCACCAA-3′
hENT2(anti-sense)5′-CCTGCGATGCTGGACTTGACCT-3′
hCNT1(sense)5′-ACCTCATAGAAGCAGCCAGC-3′
hCNT1(anti-sense)5′-CCATCAAGAAGGAGGGCTACAGGC-3′
hCNT2(sense)5′-TTTAGACACAGCCCAAAGGC-3′
hCNT2(anti-sense)5′-CCAAAGACAAAACTGGAGCC-3′
hCNT3(sense)5′-GGGTCCCTAGGAATCGTGATC-3′
hCNT3(anti-sense)5′-GAGGCGATATCACGCTTTC-3′
CD133(sense)5′-CAACAGACATGGCAACAAGG-3′
CD133(anti-sense)5′-TTCTGGATGGTGAAGTTGGC-3′
CD34(sense)5′-AATGAGGCCACAACAAACATCACA-3′
CD34(anti-sense)5′-CTGTCCTTCTTAAACTCCGCACAGC-3′
Oct-4(sense)5′-CTTGCTGCAGAAGTGGGTGGAGGAA-3′
Oct-4(anti-sense)5′-CTGCAGTGTGGGTTTCGGGCA-3′
Tie-2(sense)5′-GCCTTAATGAACCAGCACCAGG-3′
Tie-2(anti-sense)5′-ACTTCTGGGCTTCACATCTCCG -3′
KDR(sense)5′-AGACCAAAGGGGCACGATTC-3′
KDR(anti-sense)5′-GTCTGGTCTTTTGGTGTTTTGCTGT-3′
Lox-1(sense)5′- GGCTGCTGCGACTCTAGG-3′
Lox-1(anti-sense)5′-AGTGGGGCATCAAAGGAG-3′
β-Actin(sense)5′-TCAAGAACGAAAGCTGGAGG-3′
β-Actin(anti-sense)5′-GGACATCTAAGGGCATCACA-3′
18S(sense)5′-TCAAGAACGAAAGCTGGAGG-3′
18S(anti-sense)5′-GGACATCTAAGGGCATCACA-3′

Quantitative real time RT-PCR was used for hENT1 and hENT2 expression. Experiments were performed using a LightCycler™ rapid thermal cycler (Roche Diagnostics, Lewes, UK) as described [21]. In brief, reactions in 10 μL volume included 0.5 μM primers, and dNTPs, Taq DNA polymerase and reaction buffer provided in the QuantiTect SYBR Green PCR Master Mix (Qiagen, Crawley, UK). HotStart Taq DNA polymerase was activated (15 min, 95 °C), and assays included a 95 °C denaturation (15 s), annealing (20 s) at 58 °C (hENT1), 57 °C (hENT2), 56 °C (18S), and extension at 72 °C (hENT1, 15 s; hENT2, 23 s; 18S, 10 s). Fluorescent product was detected after 3-s step to 5 °C below the product melting temperature (Tm). Product specificity was confirmed by agarose gel electrophoresis (2% v/v) and melting curve analysis. The product Tm values were 79.5 °C for hENT1, 85.5 °C for hENT2 and 82.4 °C for 18S. hENT1, hENT2 and 18S standards were prepared as described [21] and oligonucleotide primers for hENT1 and hENT2 were the same as in non-quantitative RT-PCR. The number of copies for 18S mRNA was not significantly altered (P > 0.05, n = 6) in all experimental conditions used in this study (data not shown).

2.9. Statistical analysis 

Values are mean ± SEM, where n indicates number of different cell cultures (3–4 replicates). Comparisons between two and more groups were performed by means of Student’s unpaired t-test and analysis of variance (ANOVA), respectively. If the ANOVA demonstrated a significant interaction between variables, post hoc analyses were performed by the multiple-comparison Bonferroni correction test. The statistical software GraphPad Instat 3.0b and Graphpad Prism 5.0b (GraphPad Software Inc., San Diego, CA, USA) were used for data analysis. P < 0.05 was considered statistically significant.

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3. Results 

3.1. Characterization of human endothelial progenitor cells 

After 3 or 14 days of culture hEPC cells exhibited an elongated and in spindle shape (not shown), as previously reported for this cell type [2], [9], [18]. The CD133, CD34 and Oct-4 mRNA were amplified in hEPC cultured for 3 days, but not in cells cultured for 14 days (Fig. 1). On the contrary, Tie-2, KDR and Lox-1 mRNA levels were higher in hEPC cultured for 14 days compared with the minimal levels amplified in cells cultured for 3 days. Parallel experiments show that fractions of CD34+KDR− and CD34+KDR+ cells at 3 days in culture were higher (7.5 ± 0.4 and 6.9 ± 0.5 fold, respectively) compared with cells cultured for 14 days (Fig. 2); however, a larger fraction of CD34−KDR+ cells (2.7 ± 0.2 fold) was identified after 14 days compared with 3 days of culture.

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  • Fig. 1 

    Expression of non-differentiated or differentiated endothelial cell lineage makers in human endothelial progenitor cells. (A) RT-PCR for mRNA extracted from cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d). The mRNA was reversed-transcribed into cDNA, and PCRs were performed by using sequence-specific oligonucleotide primers for CD133, CD34, Oct-4, Tie-2, KDR and Lox-1, with β-actin as internal reference. Data are representative of 5 different cell cultures. (B) Gene/β-actin ratio densitometries from data in A for hEPC-3d (□) and hEPC-14d (▪). *P < 0.04 and versus corresponding values in hEPC-3d cells. Values are mean ± S.E.M. (n = 5).

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  • Fig. 2 

    Antigen expression by peripheral human endothelial progenitor cells. (A) Flow cytometry was used to identify human phycoerytrin conjugated CD34 (CD34 PE) and CarboxyFluorescein conjugated kinase insert domain receptor (KDR-FITC) antigens in human mononuclear-free cell fraction isolated from peripheral venous blood from healthy volunteers and cultured for 3 days (□) or 14 days (▪). CD34+/KDR−, CD34−/KDR+ or CD34+/KDR+ cell populations were identified. (B) CD34+/KDR−, CD34−/KDR+ or CD34+/KDR+ cell populations presented as a percentage of total number of cells. *P < 0.05 versus corresponding values in cells 3 days in culture. †P < 0.05 versus corresponding values in CD34−/KDR+ and CD34+/KDR+ cells. ‡P < 0.05 versus corresponding values in CD34+/KDR+ cells. Values are mean ± S.E.M. (n = 4).

3.2. Adenosine transport 

Overall transport of 10 μM adenosine was higher (3.8 ± 0.5 fold) in hEPC-14d compared with hEPC-3d cells (Fig. 3). Overall adenosine transport was reduced (73 ± 5%) in hECP-14d cells incubated in Krebs without sodium. However, it was unaltered in hECP-3d cells. However, adenosine transport was abolished when experiments were performed at 4 °C in Krebs with or without sodium in both cell types (not shown). Adenosine transport was also inhibited by 1 μM NBTI in hEPC-3d, but not in hEPC-14d cells. NBTI (up to 10 μM) did not significantly alter adenosine transport in hEPC-14d cells (not shown).

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  • Fig. 3 

    Adenosine transport in human endothelial progenitor cell. Overall adenosine transport (10 μM adenosine, 4 μCi/mL [3H]adenosine, 20 s, 22 °C) was measured in human endothelial progenitor cells (hEPCs) exposed to Krebs solution with sodium (NaCl) or where Na+ was equimolarly replaced by N-methylglucamine-HCl (NMG). Transport assay was performed in cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d) preincubated in Krebs (30 min) without (□) or with (▪) 1 μM nitrobenzylthioinosine (NBTI, ). *P < 0.05 versus corresponding NBTI values in hEPC-3d cells, and versus values in NMG in hEPC-14d cells. Values are mean ± S.E.M. (n = 6–8).

3.3. Kinetics of adenosine transport 

Initial transport rate of 10 μM adenosine was linear for at least 60 s in hEPC-3d and hEPC-14d cells (not shown), therefore all transport experiments were performed at 20 s of incubation. Overall transport rates for adenosine in hEPC-3d cells in absence of NBTI was semi-saturable and fitted best by the Michaelis–Menten equation plus a non-saturable, linear component for the range of concentrations used in this study (Fig. 4A). Remaining adenosine uptake in the presence of NBTI was linear in this cell type. NBTI-sensitive adenosine transport was saturable (Fig. 4B) and the Eadie–Hofstee analyses of the data were best fitted by a first order linear regression (Fig. 4C). The Vmax and apparent Km values for adenosine transport in absence of extracellular Na+ were not significantly altered compared with cells exposed to Krebs containing Na+ in hEPC-3d cells (Table 2).

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  • Fig. 4 

    Kinetics of adenosine transport in human endothelial progenitor cells 3 days in culture. (A) Overall adenosine transport (0.4–32 μM adenosine, 4 μCi/mL [3H]adenosine, 20 s, 22 °C) in Krebs without (○) or with (●) S-(4-nitrobenzyl)-6-thioinosine (0.1 μM, 30 min). (B) hENT1-mediated adenosine transport derived from data in A (see Methods). (C) Eadie–Hofstee transformation of transport data from B. V is adenosine transport rates. Values are mean ± S.E.M. (n = 7).

Table 2. Kinetic parameters for adenosine transport in human progenitor endothelial cells.
CellsConditionKm (μM)Vmax (pmol/μg protein/minute)Vmax/Km (pmol/μg protein/minute/μM)
hEPC-3dNaCl1.8 ± 0.30.028 ± 0.0010.016 ± 0.003
NMG1.6 ± 0.20.032 ± 0.0020.020 ± 0.004
hEPC-14dNa+-dependent46 ± 80.180 ± 0.0170.0039 ± 0.001
Na+-independent67 ± 160.066 ± 0.019*0.0009 ± 0.001*

Adenosine transport (0.4–32 μM adenosine, 4 μCi/ml [3H]adenosine, 20 s, 22 °C) was measured in human progenitor endothelial cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d) as described in Methods. Transport measurements were assayed in Krebs solution containing NaCl (NaCl) or in Krebs where extracellular Na+ was replaced by equimolar concentrations of N-methylglucamine-HCl (NMG). In hEPC-14d cells, the fraction of overall adenosine transport that is inhibited in Krebs without Na+ is referred as Na+-dependent transport, and remaining adenosine transport in Krebs without Na+ is referred as Na+-independent transport (see Methods). Values are means ± S.E.M. (n = 9). *P < 0.05 versus Na+-dependent transport.

In hEPC-14d cells, a semi-saturable adenosine transport that was reduced in absence of extracellular Na+, but not significantly altered by NBTI, was observed (Fig. 5A). Eadie–Hofstee analyses of overall transport data in the presence of extracellular Na+ in this cell type were well fitted by a one-phase exponential decay equation describing a biphasic curve (Fig. 5B), where at least two transport components, i.e., high affinity (apparent Km = 20 ± 5 μM), low capacity component 1, and low affinity (apparent Km = 509 ± 125 μM), high capacity component 2 were identified. In absence of Na+, component 1 was unaltered, but transport via component 2 was undetectable. Further analysis of overall transport data showed Na+-dependent and Na+-independent saturable adenosine transport activity (Fig. 5C, Table 2), both of which were best fitted by a single Michaelis–Menten equation and were lineal following Eadie–Hofstee transformation (Fig. 5D). The Na+-dependency of adenosine transport was saturable (apparent Km = 3.3 ± 0.2 mM Na+, Vmax = 0.17 ± 0.04 pmol adenosine/μg protein/minute) (Fig. 5E) and lineal in a Hill transformation of data (Hill coefficient = 1.8 ± 0.1) (Fig. 5F) in hEPC-14d cells.

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  • Fig. 5 

    Kinetics of adenosine transport in human endothelial progenitor cells 14 days in culture. (A) Overall adenosine transport (0.4–32 μM adenosine, 4 μCi/mL [3H]adenosine, 20 s, 22 °C) in Krebs with (○, ●) or without (□, ▪) sodium in absence (○, □) or presence (●, ▪) of S-(4-nitrobenzyl)-6-thioinosine (NBTI, 1 μM, 30 min). (B) Eadie-Hofstee transformation for overall adenosine transport in Krebs containing sodium in absence of NBTI as in A. Component 1 (C1) and component 2 (C2) derived from overall adenosine transport are represented (see Methods). V is adenosine transport rates. (C) Adenosine transport mediated by a Na+-dependent (○) and Na+-independent (□) component derived from data in A. (D) Eadie-Hofstee transformation of data in C. (E) Adenosine (10 μM) transport as in A in Krebs containing increasing concentrations of extracellular sodium. (F) Hill plot of transport data in C where v is initial rate and Vmax is maximal velocity of adenosine transport at varying concentrations of sodium. Values are mean ± S.E.M. (n = 7).

3.4. Specificity of adenosine transport 

Inosine inhibited adenosine transport in hEPC-3d cells (Ki = 12 ± 2 μM, n = 4), but did not significantly (P > 0.05, n = 4) alter transport in hEPC-14d cells. The nucleobases hypoxanthine and guanine at final concentrations up to 5 mM did not significantly (P > 0.05, n = 4) alter adenosine transport in both cell types (data not shown).

3.5. hENTs and hCNTs expression 

hENT1, but not hENT2 protein was detectable in hEPC-3d and hEPC-14 cells (Fig. 6A). hENT1 protein abundance was higher (6.8 ± 0.7 fold) in hEPC-3d cells compared with hEPC-14d cells. Parallel experiments show that hENT1 mRNA expression was also significantly higher (2.7 ± 0.6 fold) in hEPC-3d cells compared with hEPC-14d cells, but hENT2 mRNA was undetectable in hEPC cells (Fig. 6B). In addition, hCNT3 mRNA was undetectable in hEPC-3d cells and highly expressed in hEPC-14d cells, but hCNT1 and hCNT2 mRNA was not detected in both cell types (Fig. 6C).

  • View full-size image.
  • Fig. 6 

    hENT1 and hENT2 expression in human endothelial progenitor cells. (A) hENT1 and hENT2 protein abundance in human endothelial progenitor cells 3 days (hEPC-3d) or 14 days (hEPC-14d) in culture. Upper panel: western blot (representative of other 3 experiments) for hENT1, hENT2, and β-actin (internal reference). Protein extracts from primary cultures of human umbilical vein endothelial cells (HUVEC) was used as positive control. Lower panel: densitometric ratios for hENT1/β-actin (□) or hENT2/β-actin (▪) protein abundance. (B) Expression of hENT1 (□) or hENT2 (▪) mRNA in number of copies versus 18S rRNA (internal reference) in hEPCs as in A. (C) Relative levels of hCNT1, hCNT2 and hCNT3 mRNA in hEPC-3d and hEPC-14d cells. Upper panel: real time PCR (representative of other 3 experiments) for hCNT1, hCNT2 and hCNT3. β-Actin was internal reference. mRNA extracts from MDA148 (human breast cancer cell line), CACO-2 (human epithelial colorectal adenocarcinoma cell line) and DU-145 (human prostate cancer-derived cell line) were used as positive controls for hCNT1, hCNT2 and hCNT3, respectively. Lower panel: relative mRNA levels for hCNT1, hCNT2 and hCNT3 in hEPCs versus corresponding controls. *P < 0.05 versus all other values in hEPCs. Values are mean ± S.E.M. (n = 3–7).

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4. Discussion 

This study establishes that hEPCs express functional nucleoside membrane transporters. The inhibitor of equilibrative nucleoside transporters NBTI blocked adenosine transport in hEPC-3d cells, but this inhibitor did not significantly alter transport in hEPC-14d cells. hENT1 mRNA expression and protein abundance were reduced in hEPC-14d compared with hEPC-3d cells, a finding paralleled by increased expression of hCNT3 mRNA and endothelial cell lineage markers. In hEPC-3d cells, hENT1-mediated adenosine transport was saturable, Na+-independent and conformed by a single transport component inhibited by inosine, but unaltered by hypoxanthine or guanine. However, in hEPC-14d cells adenosine transport was semi-saturable, unaltered by NBTI, inosine, hypoxanthine or guanine, and mediated by at least two transport components, i.e., Na+-independent and Na+-dependent of high and low affinity, respectively. The Na+-dependent transport component showed a Na+:adenosine stequiometry close to 2. These findings suggest that adenosine transport is more likely mediated by hENT1 in non-differentiated hEPCs, and that a reduced hENT1-, but increased hCNT3-mediated transport activity may be associated with hEPCs differentiation to an endothelial cell phenotype. In addition, these findings could be of physiological relevance since adenosine via activation of adenosine receptors induces EPCs differentiation into endothelium [15], a phenomenon that could well depend on the modulation of extracellular concentrations of this nucleoside by nucleoside membrane transporters in the human feto-placental circulation.

The EPC nature of the cell population used in this study was confirmed by expression of the non-differentiated endothelial cell lineage makers CD133, Oct-4 and CD34 [2], [18], [19] at 3 days, but not at 14 days in culture, followed by the expression of the differentiated endothelial cell lineage makers Tie-2, KDR and Lox-1 [2] at 14 days, but not 3 days in culture. A large fraction of cells cultured for 3 days were CD34+KDR−, but after 14 days cells were mainly CD34−KDR+. These findings agree with the known temporality of CD34 (a maker for endothelial progenitor cells) [2] and Tie-2 (an endothelial cell-specific receptor) [2] surface markers expression during the phenomenon of blood vessels maturation in humans [2], [23], and suggest that cells isolated from peripheral blood used in this study are likely to be non-differentiated (hEPC-3d) and differentiated (hEPC-14d) endothelial progenitor cells.

4.1. Adenosine transport in hEPC-3d cells 

Two unrelated families of membrane transporters mediate nucleoside transport in mammalian cells, i.e., concentrative (CNTs) and equilibrative (ENTs) nucleoside membrane transporters [6], [7], [11], [24]. ENTs are Na+-independent, and according to its sensitivity to the nucleoside transport inhibitor NBTI, transport is defined as sensitive (es or ENT1) to nM NBTI or insensitive (ei or ENT2) to nM, but inhibited by μM concentrations of NBTI or by nucleobases such as hypoxanthine [6], [7], [11], [21], [24]. Our results show that hEPC-3d cells take up adenosine via a mechanism that is Na+-independent (relative contribution of Na+ to adenosine transport (1/(Na/NMG)3dF) ∼1.2; i.e., not significant from 1) (see Table 3), blocked by NBTI (Ki ∼0.6 nM) and inosine (Ki ∼12 μM), but unaffected by the nucleobases hypoxanthine or guanine, suggesting the involvement of ENT1-mediated transport activity [24]. In addition, transport was saturable with apparent Km values close to those reported for ENT1-like transport activity in most mammalian cells [6], [7], [11], including bovine adrenal medulla endothelial cells (BAdMEC, Km ∼10 μM) [25], mouse cerebral vessels (Km ∼10 μM) [26], the smooth muscle cell line DDT1 MF-2 derived from Syrian hamster myosarcoma (Km ∼9.5 μM), and the rat heart HL-1 myoblast cell line (Km ∼10 μM) [27]. However, apparent Km value in hEPC-3d cells was lower compared with primary cultures of HUVEC (Km ∼80 μM) [21], human placenta microvascular endothelium (hPMEC, Km ∼80 μM) [8], human umbilical artery smooth muscle cells (HUASMC, Km ∼190 μM) [28], or primary human cardiac microvascular endothelial cells (HCMEC, Km ∼42 μM) [29]. These differences between the Km values detected in hEPC-3d cells and other cell types may be related to inherent characteristics of cell sources, species differences, the differentiation state of the cells or the use of freshly isolated cells versus cell lines [6], [7], [8]. Interestingly, the maximal transport capacity (i.e., Vmax/Km) [6], [7], [8] in hEPC-3d cells [∼0.02 pmol/μg protein/minute/μM] was lower compared with primary cultures of HUVEC [1–3 pmol/μg protein/minute/μM] [6], [7], [21], ∼10-fold higher than values reported in primary cultures of hPMEC [0.002–0.003 pmol/μg protein/minute/μM] [8]; however, within the range of values reported in BAdMEC [∼0.09 pmol/μg protein/minute/μM] [25], HUASMC [0.09–0.1 pmol/μg protein/minute/μM] [28] and HCMEC [∼0.22 pmol/μg protein/minute/μM] [29]. Since hEPC-3d cells are apparently still in a process of maturation, it is feasible that variability of ENT1-like transport activity detected in this cell type could also reflect its non-differentiated stage compared with differentiated endothelial cells from the human macro (HUVEC) and microvasculature (hPMEC).

Table 3. Relative contribution of sodium to adenosine transport in human progenitor endothelial cells.
C1/C2-NaF0.9 ± 0.1
C1/C2-NMGF>>1*
1/(Na/NMG)3dF1.2 ± 0.2
1/(Na/NMG)14dFC10.8 ± 0.1
1/(Na/NMG)14dFC2>>1*

The maximal velocity (Vmax) and apparent Michaelis–Menten constant (Km) were derived from overall adenosine transport (0.4–32 μM adenosine, 4 μCi/mL [3H]adenosine, 20 s, 22 °C) in human progenitor endothelial cells cultured for 3 days (hEPC-3d) or 14 days (hEPC-14d) (see Table 2) as described in Methods. Transport measurements were assayed in Krebs solution containing NaCl (Na) or in Krebs where extracellular Na+ was replaced by equimolar concentrations of N-methylglucamine-HCl (NMG). Component 1 (C1) and component 2 (C2) of transport were derived from the Eadie-Hofstee analysis of overall adenosine transport in Krebs containing Na+ in hEPC-14d cells (see Methods). The relative contribution of components 1 and 2 in hEPC-14d cells to overall adenosine transport in the presence of Na+ or NMG was stimated by C1/C2-NaF or C1/C2-NMGF, respectively (see Methods). The relative contribution of Na+ to adenosine transport via components 1 or 2 in hEPC-14d cells was estimated by 1/(Na/NMG)14dFC1 or 1/(Na/NMG)14dFC2, respectively. The relative contribution of Na+ to the unique transport component in hEPC-3d cells was estimated by 1/(Na/NMG)3dF. Values are means ± S.E.M. (n = 6). * is significantly different from 1 which corresponds to a comparable contribution of the conditions to the phenomenon (i.e., control). Expression >>1 denotes infinite higher than 1 by absence of Na+-dependent transport component.

4.2. Adenosine transport in hEPC-14d cells 

Overall adenosine transport in hEPC-14d cells exhibit at least two components, i.e., component 1 of high affinity and low capacity, and component 2 of low affinity and high capacity, both of which exhibited similar maximal transport capacity (Vmax/Km ∼0.0012 pmol//μg protein/minute/μM) and relative contribution to total adenosine transport in the presence of Na+ (C1/C2-NaF ∼0.9; i.e., not significant from 1). Since component 2 was undetectable in absence of Na+ (C1/C2-NMGF > >1 and 1/(Na/NMG)14dFC2 > >1) and unaltered by NBTI, and component 1 was Na+-independent (1/(Na/NMG)14dFC1 ∼0.8; i.e., not significant from 1) and unaltered by NBTI, it is feasible that at least CNTs-like mediated transport most likely representing component 2 is functional in this cell type. In addition to the CNTs-like transport, component 1 was due to the activity of a non-classical equilibrative, non-concentrative nucleoside transport (ncECNT) yet to be identified. Our results show that hENT1 protein abundance in hEPC-14d cells is significantly, but not totally reduced compared with hEPC-3d cells, thus a remaining hENT1-mediated transport in hEPC-14d cells could be feasible. However, overall adenosine transport in hEPC-14d cells exposed to 1 μM NBTI was lower, although nonsignificant, thus making unlikely this possibility. In addition, since hENT1 protein abundance was determined in whole cell extracts, we can not assure that remaining hENT1 will be in fact active or located at the plasma membrane in this cell type. Studies in microvascular endothelial cells (MVECs) derived from rat skeletal muscle show that these cells coexpress ENT1 and ENT2 membrane transporters, as well as a lower, but significant purine-selective, Na+-dependent CNT2-like transport activity [30]. In addition, in the immortalized rat brain endothelial cell line RBE4 [31] and in primary cultures of rat brain endothelial cells (RBEC) [32], ENT1, ENT2, CNT2 and CNT3 nucleoside transporters are coexpressed. However, endothelial cells from large vessels such as HUVEC express mainly, if not only, ENT1, ENT2 and ENT4-subtype nucleoside transporters [6], [7], [18], [33]. In this study, hCNT1 and hCNT2 are not expressed, but hCNT3 mRNA is detectable in parallel to a nucleoside transport activity with a stequiometry of ∼2, which is classical characteristic for CNT3-mediated nucleoside transport [10], [11]. Thus, expression of ENTs and CNTs may be differential in hEPC-3d compared with hEPC-14d cells, most likely playing a differential role regarding their contribution in the removal of extracellular adenosine and modulation of adenosine biological actions [4], [5], [6], [7], [24]. Since changes in adenosine transport detected in these cell types reflect the actual state only at 3 or 14 days of culture, not a conclusion on the role of nucleoside transporters expression on EPCs differentiation can be stated. Interestingly, preliminary observations suggest that adenosine receptor subtypes A2A, A2B and A3, but not A1 mRNA expression, is detectable in hEPC-3d and hEPC-14d cells [34]. Pattern of expression (i.e., A2A > A2B > A3) was similar in both cell types, suggesting instead that this is a phenomenon that could not be associated with hEPCs differentiation, as proposed to be for adenosine transporters expression.

Results on kinetics of adenosine transport in hEPC-14d cells show that the relative contribution of component 1 (i.e., ncECNT-like) versus component 2 (i.e., CNTs-like) to total adenosine transport in the presence of Na+ is similar (C1/C2-NaF ∼0.9), suggesting the possibility that ncECNTs-like transport and CNTs-like transport activity contribution to total transport is equivalent in this cell type. Furthermore, the Vmax/Km and the relative contribution of Na+ to adenosine transport in hEPC-3d cells, as well as component 1 of transport in hEPC-14d cells, were not significantly altered by the removal of this ion from the extracellular medium. Thus, the possibility that expression of CNTs-like compared to other nucleoside transport mechanism(s) is differential in hEPCs, are similar to reports in cardiac fibroblasts from streptozotocin-induced diabetic rats where unidirectional Na+-dependent adenosine uptake and CNT1 and CNT2 isoforms expression are increased in parallel with reduced ENTs-like transport and ENT1 and ENT2 expression [35]. Thus, it is likely that regulation of ENTs and CNTs expression in hEPCs could also respond to differential modulation depending on the pathophysiological state.

A reduced ENT1 expression, limiting ENTs-like activity, but increased Na+-dependent nucleoside transport in cell differentiation has also been reported in trans-retinoic acid stimulated NB4 cells (a cell line derived from acute myeloid leukaemia) [36]. In addition, ENT1-like transport activity is lost during maturation of adult sheep reticulocytes into erythrocytes [37], and ENT1- and ENT2-mediated nucleoside transport activity is decreased in parallel with increased Na+-dependent uridine transport following human promyelocytic HL-60 leukaemia cells differentiation along either the granulocytic or the monocytic pathway [38]. Thus, ENTs- and CNTs-like adenosine transport could be selectively modulated by or leading to hEPC differentiation. This possibility is supported by the findings showing that hENT1 expression in hEPC-14d cells is negligible compared with hEPC-3d cells, and that hCNT3 expression is predominant in hEPC-14d cells only. These findings also suggest a potential link between reduction in ENT1- and increased CNT3-like transport activity and hEPCs differentiation. Since adenosine modulates key endothelial cell functions, such as synthesis of nitric oxide and l-arginine metabolism [6], [7], we hypothesize that differential expression of hENTs and hCNTs in hEPCs is a phenomenon that could be determinant in the endothelium-dependent modulation of blood flow in mature vessels in health and in disease, including pathological conditions associated with altered plasma adenosine levels due, at least in part, to abnormal hENTs-mediated adenosine transport such as in hypoxia, gestational diabetes or pre-eclampsia [6], [7], [8], [21]. In addition, it is known that adenosine acting via adenosine receptors induces EPCs recruitment to sites of neovascularization where they differentiate into endothelial cells [15], and impaired movilization of EPCs derived from the mother into systemic circulation has been associated with insufficient regeneration of endothelial cells in the utero-placental circulation in pre-eclampsia [3], a disease characterized by abnormal placenta vascularization [39]. Thus, it is feasible that acute modulation of extracellular adenosine concentration by nucleoside membrane transporters in vascular beds where tone is modulated by locally-released factors such as the feto-placental circulation [8], [39], is a phenomenon that could perhaps play key roles in EPCs differentiation (at least for 3 versus 14 days in culture) to endothelial cells, therefore modulating its several biological effects.

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Acknowledgments 

Fondo Nacional de Desarrollo Científico y Tecnológico (FONDECYT 11070035, 1070865, 1080534), Dirección de Investigación, Universidad de Concepción (DIUC 205-072.031-1.0), and Programa de Investigación Interdisciplinario (PIA) from Comisión Nacional de Investigación en Ciencia y Tecnología (CONICYT)(Anillos ACT-73), Chile. E Guzmán-Gutiérrez and B Krause hold CONICYT-PhD (Chile) fellowships. E Guzmán-Gutiérrez was the recipient of a Postgraduate School, Universidad de Concepción-MSc (Chile) fellowship. C Salomón holds a Faculty of Medicine, Pontificia Universidad Católica de Chile-PhD fellowship.

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Appendix. Supplementary data 

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References 

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PII: S0143-4004(10)00285-7

doi:10.1016/j.placenta.2010.07.016

Placenta
Volume 31, Issue 10 , Pages 928-936, October 2010

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