Differential expression of functional nucleoside transporters in non-differentiated and differentiated human endothelial progenitor cells.
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.
Guzmán-Gutiérrez
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Department of Clinical Biochemistry and Immunology, Faculty of Pharmacy, Universidad de Concepción, Chile.
Sandoval
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C
Nova
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E
Castillo
J L
JL
Vera
J C
JC
Lamperti
L
L
Krause
B
B
Salomón
C
C
Sepúlveda
C
C
Aguayo
C
C
Sobrevia
L
L
D000241:Adenosine / Q000378:metabolism; D001692:Biological Transport; D015153:Blotting, Western; D002454:Cell Differentiation / Q000502:physiology; D042783:Endothelial Cells / Q000378:metabolism / Q000502:physiology*; D033721:Equilibrative Nucleoside Transporter 1 / Q000096:biosynthesis* / Q000235:genetics; D033722:Equilibrative-Nucleoside Transporter 2 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007700:Kinetics; D012313:RNA / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000378:metabolism / Q000502:physiology*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology
D033721:Equilibrative Nucleoside Transporter 1; D033722:Equilibrative-Nucleoside Transporter 2; D013868:Thioinosine; D012313:RNA; C001789:4-nitrobenzylthioinosine; D000241:Adenosine